Respiratory syncytial virus vaccines expressing protective antigens from promoter-proximal genes

ABSTRACT

Recombinant respiratory syncytial virus (RSV) having the position of genes shifted within the genome or antigenome of the recombinant virus are infectious and attenuated in humans and other mammals. Gene shifted RSV are constructed by insertion, deletion or rearrangement of genes or genome segments within the recombinant genome or antigenome and are useful in vaccine formulations for eliciting an anti-RSV immune response. Also provided are isolated polynucleotide molecules and vectors incorporating a recombinant RSV genome or antigenome wherein a gene or gene segment is shifted to a more promoter-proximal or promoter-distal position within the genome or antigenome compared to a wild type position of the gene in the RSV gene map. Shifting the position of genes in this manner provides for a selected increase or decrease in expression of the gene, depending on the nature and degree of the positional shift. In one embodiment, RSV glycoproteins are upregulated by shifting one or more glycoprotein-encoding genes to a more promoter-proximal position. Genes of interest for manipulation to create gene position-shifted RSV include any of the NS1, NS2, N, P, M, SH, M2(ORF1), M2(ORF2), L, F or G genes or a genome segment that may be part of a gene or extragenic. A variety of additional mutations and nucleotide modifications are provided within the gene position-shifted RSV of the invention to yield desired phenotypic and structural effects.

CROSS-REFERENCES TO RELATED APPLICATIONS

[0001] This application claims the priority benefit of U.S. ProvisionalApplication No. 60/213,708, filed by Krempl et al. on Jun. 23, 2000.

BACKGROUND OF THE INVENTION

[0002] Human respiratory syncytial virus (HRSV) is the leading viralagent of serious pediatric respiratory tract disease worldwide (Collins,et al., Fields Virology 2:1313-1352, 1996). RSV outranks all othermicrobial pathogens as a cause of pneumonia and bronchiolitis in infantsunder one year of age. Virtually all children are infected by two yearsof age, and reinfection occurs with appreciable frequency in olderchildren and young adults (Chanock et al., in Viral Infections ofHumans, 3rd ed., A. S. Evans, ed., Plenum Press, N.Y., 1989). RSV isresponsible for more than one in five pediatric hospital admissions dueto respiratory tract disease, and in the United States alone causesnearly 100,000 hospitalizations and 4,500 deaths yearly. (Heilman, JInfect Dis 161:402-6, 1990). In addition, there is evidence that seriousrespiratory tract infection early in life can initiate or exacerbateasthma (Sigurs, et al., Pediatrics 95:500-5, 1995).

[0003] While human RSV usually is thought of in the context of thepediatric population, it also is recognized as an important agent ofserious disease in the elderly (Falsey, et al., J. Infect. Dis.172:389-394, 1995). Human RSV also causes life-threatening disease incertain immunocompromised individuals, such as bone marrow transplantrecipients (Fouillard, et al., Bone Marrow Transplant 9:97-100, 1992).

[0004] For treatment of human RSV, one chemotherapeutic agent,ribavirin, is available. However, its efficacy and use is controversial.There are also licensed products for RSV intervention which are composedof pooled donor lgG (Groothuis, et al., N Engl J Med 329:1524-30, 1993)or a humanized RSV-specific monoclonal antibody. These are administeredas passive immunoprophylaxis agents to high risk individuals. Whilethese products are useful, their high cost and other factors, such aslack of long term effectiveness, make them inappropriate for widespreaduse. Other disadvantages include the possibility of transmittingblood-borne viruses and the difficulty and expense in preparation andstorage. Moreover, the history of the control of infectious diseases,and especially diseases of viral origin, indicates the primaryimportance of vaccines.

[0005] Despite decades of investigation to develop effective vaccineagents against RSV, no safe and effective vaccine has yet been achievedto prevent the severe morbidity and significant mortality associatedwith RSV infection. Failure to develop successful vaccines relates inpart to the fact that small infants have diminished serum and secretoryantibody responses to RSV antigens. Thus, these individuals suffer moresevere infections from RSV, whereas cumulative immunity appears toprotect older children and adults against more serious impacts of thevirus.

[0006] The mechanisms of immunity in RSV infection have recently comeinto focus. Secretory antibodies appear to be most important inprotecting the upper respiratory tract, whereas high levels of serumantibodies are thought to have a major role in resistance to RSVinfection in the lower respiratory tract. RSV-specific cytotoxic Tcells, another effector arm of induced immunity, are also important inresolving an RSV infection. However, while this latter effector can beaugmented by prior immunization to yield increased resistance to viruschallenge, the effect is short-lived. The F and G surface glycoproteinsare the two major protective antigens of RSV, and are the only two RSVproteins which have been shown to induce RSV neutralizing antibodies andlong term resistance to challenge (Collins et al., Fields Virology,Fields et al., eds., 2:1313- 1352, Lippincott-Raven, Philadelphia, 1996;Connors et al., J. Virol. 65(3):1634-7, 1991). The third RSV surfaceprotein, SH, did not induce RSV-neutralizing antibodies or significantresistance to RSV challenge.

[0007] An obstacle to developing live RSV vaccines is the difficulty inachieving an appropriate balance between attenuation and immunogenicity,partly due to the genetic instability of some attenuated viruses, therelatively poor growth of RSV in cell culture, and the instability ofthe virus particle. In addition the immunity which is induced by naturalinfection is not fully protective against subsequent infection. A numberof factors probably contribute to this, including the relativeinefficiency of the immune system in restricting virus infection on theluminal surface of the respiratory tract, the short-lived nature oflocal mucosal immunity, rapid and extensive virus replication, reducedimmune responses in the young due to immunological immaturity,immunosuppression by transplacentally derived maternal serum antibodies,and certain features of the virus such as a high degree of glycosylationof the G protein. Also, as will be described below, human RSV exists astwo antigenic subgroups A and B, and immunity against one subgroup is ofreduced effectiveness against the other.

[0008] Although RSV can reinfect multiple times during life,reinfections usually are reduced in severity due to protective immunityinduced by prior infection, and thus immunoprophylaxis is feasible. Alive-attenuated RSV vaccine would be administered intranasally toinitiate a mild immunizing infection. This has the advantage ofsimplicity and safety compared to a parenteral route. It also providesdirect stimulation of local respiratory tract immunity, which plays amajor role in resistance to RSV. It also abrogates the immunosuppressiveeffects of RSV-specific maternally-derived serum antibodies, whichtypically are found in the very young. Also, while the parenteraladministration of RSV antigens can sometimes be associated withimmunopathologic complications (Murphy et al., Vaccine 8(5):497-502,1990), this has never been observed with a live virus.

[0009] A formalin-inactivated virus vaccine was tested against RSV inthe mid-1960s, but failed to protect against RSV infection or disease,and in fact exacerbated symptoms during subsequent infection by thevirus. (Kim et al., Am. J. Epidemiol., 89:422-434, 1969; Chin et al., AmJ. Epidemiol., 89:449-463, 1969; Kapikian et al., Am. J. Epidemiol.,89:405-421, 1969).

[0010] More recently, vaccine development for RSV has focused onattenuated RSV mutants. Friedewald et al., (J. Amer. Med. Assoc.204:690-694, 1968) reported a cold passaged mutant of RSV (cpRSV) whichappeared to be sufficiently attenuated to be a candidate vaccine. Thismutant exhibited a slight increased efficiency of growth at 26° C.compared to its wild-type (wt) parental virus, but its replication wasneither temperature sensitive nor significantly cold-adapted. Thecold-passaged mutant, however, was attenuated for adults. Althoughsatisfactorily attenuated and immunogenic for infants and children whohad been previously infected with RSV (i.e., seropositive individuals),the cpRSV mutant retained a low level virulence for the upperrespiratory tract of seronegative infants.

[0011] Similarly, Gharpure et al., (J. Virol. 3:414-421, 1969) reportedthe isolation of temperature sensitive RSV (tsRSV) mutants which alsowere promising vaccine candidates. One mutant, ts-1, was evaluatedextensively in the laboratory and in volunteers. The mutant producedasymptomatic infection in adult volunteers and conferred resistance tochallenge with wild-type virus 45 days after immunization. Again, whileseropositive infants and children underwent asymptomatic infection,seronegative infants developed signs of rhinitis and other mildsymptoms. Furthermore, instability of the ts phenotype was detected.Although virus exhibiting a partial or complete loss of temperaturesensitivity represented a small proportion of virus recoverable fromvaccinees, it was not associated with signs of disease other than mildrhinitis.

[0012] These and other studies revealed that certain cold-passaged andtemperature sensitive RSV strains were underattenuated and caused mildsymptoms of disease in some vaccinees, particularly seronegativeinfants, while others were overattenuated and failed to replicatesufficiently to elicit a protective immune response, (Wright et al.,Infect. Immun., 37:397-400, 1982). Moreover, genetic instability ofcandidate vaccine mutants has resulted in loss of their temperaturesensitive phenotype, further hindering development of effective RSVvaccines. See generally, (Hodes et al., Proc. Soc. Exp. Biol. Med.145:1158-1164, 1974; McIntosh et al., Pediatr. Res. 8:689-696, 1974; andBelshe et al., J. Med. Virol., 3:101-110, 1978).

[0013] As an alternative to live-attenuated RSV vaccines, investigatorshave also tested subunit vaccine candidates using purified RSV envelopeglycoproteins. The glycoproteins induced resistance to RS virusinfection in the lungs of cotton rats, (Walsh et al., J. Infect. Dis.155:1198--1204, 1987), but the antibodies had very weak neutralizingactivity and immunization of rodents with purified subunit vaccine ledto disease potentiation (Murphy et al., Vaccine 8:497-502, 1990).

[0014] Recombinant vaccinia virus vaccines which express the F or Genvelope glycoprotein have also been explored. These recombinantsexpress RSV glycoproteins which are indistinguishable from the authenticviral counterpart, and rodents infected intradermally with vaccinia-RSVF and G recombinants developed high levels of specific antibodies thatneutralized viral infectivity. Indeed, infection of cotton rats withvaccinia-F recombinants stimulated almost complete resistance toreplication of RSV in the lower respiratory tract and significantresistance in the upper tract. (Olmsted et al., Proc. Natl. Acad. Sci.USA 83:7462-7466, 1986). However, immunization of chimpanzees withvaccinia-F and -G recombinant provided almost no protection against RSVchallenge in the upper respiratory tract (Collins et al., Vaccine8:164-168, 1990) and inconsistent protection in the lower respiratorytract (Crowe et al., Vaccine 11: 1395-1404, 1993).

[0015] Despite these various efforts to develop an effective RSVvaccine, no licensed vaccine has yet been approved for RSV. Theunfulfilled promises of prior approaches underscores a need for newstrategies to develop RSV vaccines, and in particular methods formanipulating recombinant RSV to incorporate genetic changes that yieldnew phenotypic properties in viable, attenuated RSV recombinants.However, manipulation of the genomic RNA of RSV and other non-segmentednegative-sense RNA viruses has heretofore proven difficult. Majorobstacles in this regard include non-infectivity of naked genomic RNA ofthese viruses, poor viral growth in tissue culture, lengthy replicationcycles, virion instability, a complex genome, and a refractoryorganization of gene products.

[0016] Recombinant DNA technology has made it possible to recoverinfectious non-segmented negative-stranded RNA viruses from cDNA, togenetically manipulate viral clones to construct novel vaccinecandidates, and to rapidly evaluate their level of attenuation andphenotypic stability (for reviews, see Conzelmann, J. Gen. Virol.77:381-89, 1996; Palese et al., Proc. Natl. Acad. Sci. U.S.A.93:11354-58, 1996). In this context, recombinant rescue has beenreported for infectious respiratory syncytial virus (RSV), parainfluenzavirus (PIV), rabies virus (RaV), vesicular stomatitis virus (VSV),measles virus (MeV), rinderpest virus and Sendai virus (SeV) fromcDNA-encoded antigenomic RNA in the presence of essential viral proteins(see, e.g., Garcin et al., EMBO J. 14:6087-6094, 1995; Lawson et al.,Proc. Natl. Acad. Sci. U.S.A. 92:4477-81, 1995; Radecke et al., EMBO J.14:5773-5784, 1995; Schnell et al., EMBO J. 13:4195-203, 1994; Whelan etal., Proc. Natl. Acad. Sci. U.S.A. 92:8388-92, 1995; Hoffman et al., JVirol. 71:4272-4277, 1997; Pecters et al., J. Virol. 73:5001-5009, 1999;Kato et al., Genes to Cells 1:569-579, 1996; Roberts et al., Virology247(1), 1-6, 1998; Baron et al., J Virol. 71:1265-1271, 1997;International Publication No. WO 97/06270; U.S. Provisional PatentApplication No. 60/007,083, filed Sep. 27, 1995; U.S. patent applicationSer. No. 08/720,132, filed Sep. 27, 1996; U.S. Provisional PatentApplication No. 60/021,773, filed Jul. 15, 1996; U.S. Provisional PatentApplication No. 60/046,141, filed May 9, 1997; U.S. Provisional PatentApplication No. 60/047,634, filed May 23, 1997; U.S. Pat. No. 5,993,824,issued Nov. 30, 1999 (corresponding to International Publication No. WO98/02530); U.S. patent application Ser. No. 09/291,894, filed by Collinset al. on Apr. 13, 1999; U.S. Provisional Patent Application No.60/129,006, filed by Murphy et al. on Apr. 13, 1999; Collins, et al.,Proc Nat. Acad. Sci. U.S.A. 92:11563-11567, 1995; Bukreyev, et al., JVirol 70:6634-41, 1996, Juhasz et al., J. Virol. 71(8):5814-5819, 1997;Durbin et al., Virology 235:323-332, 1997; He et al. Virology237:249-260, 1997; Baron et al. J. Virol. 71:1265-1271, 1997; Whiteheadet al., Virology 247(2):232-9, 1998a; Buchholz et al. J. Virol.73:251-9, 1999; Whitehead et al., J. Virol. 72(5):4467-4471, 1998b; Jinet al. Virology 251:206-214, 1998; and Whitehead et al., J. Virol.73:(4)3438-3442, 1999, and Bukreyev, et al., Proc Nat Acad Sci USA96:2367-72, 1999, each incorporated herein by reference in its entiretyfor all purposes).

[0017] Based on the foregoing developments, it is now possible torecover infectious RSV from cDNA and to design and implement variousgenetic manipulations to RSV clones to construct novel vaccinecandidates. Thereafter, the level of attenuation and phenotypicstability, among other desired phenotypic characteristics, can beevaluated and adjusted. The challenge which thus remains is to develop abroad and diverse menu of genetic manipulations that can be employed,alone or in combination with other types of genetic manipulations, toconstruct infectious, attenuated RSV clones that are useful for broadvaccine use. In this context, an urgent need remains in the art foradditional tools and methods that will allow engineering of safe andeffective vaccines to alleviate the serious health problems attributableto RSV. Surprisingly, the present invention fulfills this need byproviding additional tools for constructing infectious, attenuated RSVvaccine candidates.

SUMMARY OF THE INVENTION

[0018] The present invention provides recombinant respiratory syncytialviruses (RSVs) which are modified by shifting a relative gene order orspatial position of one or more genes or genome segments within arecombinant RSV genome or antigenome—to generate a recombinant vaccinevirus that is infectious, attenuated and immunogenic in humans and othermammals. The recombinant RSVs of the invention typically comprise amajor nucleocapsid (N) protein, a nucleocapsid phosphoprotein (P), alarge polymerase protein (L), a RNA polymerase elongation factor, and apartial or complete recombinant RSV genome or antigenome having one ormore positionally shifted RSV genes or genome segments within therecombinant genome or antigenome. In certain aspects of the invention,the recombinant RSV features one or more positionally shifted genes orgenome segments that may be shifted to a more promoter-proximal orpromoter-distal position by insertion, deletion, or rearrangement of oneor more displacement polynucleotides within the partial or completerecombinant RSV genome or antigenome. Displacement polynucleotides maybe inserted or rearranged into a non-coding region (NCR) of therecombinant genome or antigenome, or may be incorporated in therecombinant RSV genome or antigenome as a separate gene unit (GU).

[0019] In exemplary embodiments of the invention, isolated infectiousrecombinant RSV are constructed by addition, deletion, or rearrangementof one or more displacement polynucleotides that may be selected fromone or more RSV gene(s) or genome segment(s) selected from RSV NS1, NS2,N, P, M, SH, M2(ORF1), M2(ORF2), L, F and G genes and genome segmentsand leader, trailer and intergenic regions of the RSV genome andsegments thereof. In more detailed embodiments, polynucleotide inserts,and deleted or rearranged elements within the recombinant RSV genome orantigenome are selected from one or more bovine RSV (BRSV) or human RSV(HRSV) gene(s) or genome segment(s) selected from RSV NS1, NS2, N, P, M,SH, M2(ORF1), M2(ORF2), L, F and G gene(s) or genome segment(s) andleader, trailer and intergenic regions of the RSV genome or segmentsthereof.

[0020] In certain aspects of the invention, displacement polynucleotidesare deleted to form the recombinant RSV genome or antigenome. Deletionof a displacement polynucleotide in this manner causes a positionalshift of one or more “shifted” RSV genes or genome segments within therecombinant genome or antigenome to a more promoter-proximal positionrelative to a position of the shifted gene(s) or genome segment(s)within a wild type RSV (e.g., HRSV A2 or BRSV kansas strain) genome orantigenome. Exemplary displacement polynucleotides that may be deletedin this manner to form the recombinant RSV genome or antigenome may beselected from one or more RSV NS1, NS2, SH, M2(ORF2), or G gene(s) orgenome segment(s) thereof.

[0021] In more detailed embodiments of the invention, a displacementpolynucleotide comprising a RSV NS1 gene is deleted to form therecombinant RSV genome or antigenome. Alternatively, a displacementpolynucleotide comprising a RSV NS2 gene may be deleted to form therecombinant RSV genome or antigenome. Alternatively, a displacementpolynucleotide comprising a RSV SH gene may be deleted to form therecombinant RSV genome or antigenome. Alternatively, a displacementpolynucleotide comprising RSV M2(ORF2) can be deleted to form therecombinant RSV genome or antigenome. Alternatively, a displacementpolynucleotide comprising a RSV G gene may be deleted to form therecombinant RSV genome or antigenome or antigenome.

[0022] In yet additional embodiments, multiple displacementpolynucleotides comprising RSV genes or genome segments may be deleted.For example, RSV F and G genes may both be deleted to form therecombinant RSV genome or antigenome or antigenome. Alternatively, theRSV NS1 and NS2 genes may both be deleted to form the recombinant RSVgenome or antigenome or antigenome. Alternatively, the RSV SH and NS2genes may both be deleted to form the recombinant RSV genome orantigenome or antigenome. Alternatively, the RSV SH, NS1 and NS2 genescan all be deleted to form the recombinant RSV genome or antigenome orantigenome.

[0023] In different embodiments of the invention, isolated infectiousrecombinant RSV are provided wherein one or more displacementpolynucleotides is/are added, substituted, or rearranged within therecombinant RSV genome or antigenome to cause a positional shift of oneor more shifted RSV gene(s) or genome segment(s). Among thesemodifications, gene and genome segment insertions and rearrangements mayintroduce or rearrange the subject genes or genome segments to a morepromoter-proximal or promoter-distal position relative to a respectiveposition of each subject (inserted or rearranged) gene or genome segmentwithin a corresponding (e.g., bovine or human) wild type RSV genome orantigenome. Displacement polynucleotides which may be added,substituted, or rearranged within the recombinant RSV genome orantigenome can be selected from one or more of the RSV NS1, NS2, SH,M2(ORF2), F, and/or G gene(s) or genome segment(s) thereof.

[0024] In more detailed embodiments, displacement polynucleotides areselected for insertion or rearrangement within the recombinant RSVgenome or antigenome which comprises one or more RSV genes or genomesegments that encoded one or more RSV glycoproteins or immunogenicdomains or epitopes of RSV glycoproteins. In exemplary embodiments,these displacement polynucleotides are selected from genes or genomesegments encoding RSV F, G, and/or SH glycoproteins or immunogenicdomains or epitopes thereof. For example, one or more RSV glycoproteingene(s) selected from F, G and SH may be added, substituted orrearranged within the recombinant RSV genome or antigenome to a positionthat is more promoter-proximal or promoter-distal compared to the wildtype gene order position of the gene(s).

[0025] In exemplary embodiments, the RSV glycoprotein gene G isrearranged within the recombinant RSV genome or antigenome to a geneorder position that is more promoter-proximal compared to the wild typegene order position of G. In more detailed aspects, the RSV glycoproteingene G is shifted to gene order position 1 within said recombinant RSVgenome or antigenome. In other exemplary embodiments, the RSVglycoprotein gene F is rearranged within the recombinant RSV genome orantigenome to a more promoter-proximal position, for example by shiftingthe F gene to gene order position 1 within the recombinant genome orantigenome. In yet additional exemplary embodiments, both RSVglycoprotein genes G and F are rearranged within the recombinant RSVgenome or antigenome to gene order positions that are morepromoter-proximal compared to their respective wild type gene orderpositions. In more detailed aspects, the RSV glycoprotein gene G isshifted to gene order position 1 and the RSV glycoprotein gene F isshifted to gene order position 2.

[0026] In yet additional constructs featuring glycoprotein gene shifts,recombinant RSV are produced having one or more RSV glycoprotein gene(s)selected from F, G and SH, or a genome segment thereof, added,substituted or rearranged within the recombinant RSV genome orantigenome, wherein one or more RSV NS1, NS2, SH, M2(ORF2), or G gene(s)or genome segment(s) thereof is/are deleted. Thus, a gene or genomesegment of RSV F, G, or SH may be added, substituted or rearranged in abackground wherein a displacement polynucleotide comprising a RSV NS1gene is deleted to form the recombinant RSV genome or antigenome.Alternatively, a gene or genome segment of RSV F, G, or SH may be added,substituted or rearranged in a background wherein a displacementpolynucleotide comprising a RSV NS2 gene is deleted to form therecombinant RSV genome or antigenome. Alternatively, a gene or genomesegment of RSV F, G, or SH may be added, substituted or rearranged in abackground wherein a displacement polynucleotide comprising a RSV SHgene is deleted to form the recombinant RSV genome or antigenome.

[0027] In one example below, the RSV glycoprotein gene G is rearrangedwithin a recombinant RSV genome or antigenome having an SH gene deletionto a gene order position that is more promoter-proximal compared to thewild type gene order position of G. In more detailed aspects, the RSVglycoprotein gene G is shifted to gene order position 1 within therecombinant RSV genome or antigenome, as exemplified by the recombinantvaccine candidate G1/ΔSH. In another example, the RSV glycoprotein geneF is rearranged within a recombinant RSV genome or antigenome having anSH gene deletion to a more promoter-proximal proximal position. In moredetailed aspects, the F gene is shifted to gene order position 1, asexemplified by the recombinant F1ΔSH. In yet another example below, bothRSV glycoprotein genes G and F are rearranged within a ΔSH recombinantRSV genome or antigenome to gene order positions that are morepromoter-proximal compared to the wild type gene order positions of Gand F. In more detailed aspects, the RSV glycoprotein gene G is shiftedto gene order position 1 and the RSV glycoprotein gene F is shifted togene order position 1 within the recombinant RSV genome or antigenome,as exemplified by the recombinant G1F2/ΔSH.

[0028] Yet additional examples of gene position-shifted RSV are providedfeaturing shifts of glycoprotein gene(s) selected from F, G and SH,which are produced within a recombinant RSV genome or antigenome havingmultiple genes or genome segments selected from RSV NS1, NS2, SH,M2(ORF2), and G gene(s) or genome segment(s) deleted. In one example,the RSV SH and NS2 genes are both deleted to form the recombinant RSVgenome or antigenome or antigenome, and one or both RSV glycoproteingenes G and F are rearranged within the recombinant RSV genome to morepromoter-proximal gene order positions. In more detailed aspects, G isshifted to gene order position 1 and F is shifted to gene order position2, as exemplified by the recombinant G1F2/ΔNS2ΔSH. In another example,all of the RSV SH, NS1 and NS2 genes are deleted to form the recombinantRSV genome or antigenome or antigenome, and one or both RSV glycoproteingenes G and F are rearranged within the recombinant RSV genome orantigenome to more promoter-proximal positions, as exemplified by therecombinant vaccine candidate G1F2/ΔNS2ΔNS2ΔSH.

[0029] In yet additional aspects of the invention, gene position-shiftedRSV are combined with or incorporated within human-bovine chimeric RSV.Within these aspects, the recombinant genome or antigenome comprises apartial or complete human RSV (HRSV) or bovine RSV (BRSV) backgroundgenome or antigenome combined with one or more heterologous gene(s) orgenome segment(s) from a different RSV to for a human-bovine chimericRSV genome or antigenome. The heterologous gene or genome segment of thedifferent, HRSV or BRSV may be added or substituted at a position thatis more promoter-proximal or promoter-distal compared to a wild typegene order position of a counterpart gene or genome segment within thepartial or complete HRSV or BRSV background genome or antigenome. In onesuch example provided herein, both human RSV glycoprotein genes G and Fare substituted at gene order positions 1 and 2, respectively, toreplace counterpart G and F glycoprotein genes deleted at wild typepositions 7 and 8, respectively in a partial bovine RSV backgroundgenome or antigenome, as exemplified by the recombinant virusrBRSV/A2-G1F2. In other embodiments, one or more human RSVenvelope-associated genes selected from F, G, SH, and M is/are added orsubstituted within a partial or complete bovine RSV background genome orantigenome. In more detailed aspects, one or more human RSVenvelope-associated genes selected from F, G, SH, and M is/are added orsubstituted within a partial bovine RSV background genome or antigenomein which one or more envelope-associated genes selected from F, G, SH,and M is/are deleted. In one example described below, human RSVenvelope-associated genes F, G, and M are added within a partial bovineRSV background genome or antigenome in which all of theenvelope-associated genes F, G, SH, and M are deleted, as exemplified bythe recombinant virus rBRSV/A2-MGF.

[0030] In another alternate embodiment of the invention, isolatedinfectious recombinant RSV are provided in which the RSV M2(ORF1) isshifted to a more promoter-proximal position within the recombinant RSVgenome or antigenome. The result of this gene shift is to upregulatetranscription of the recombinant virus.

[0031] In additional aspects of the invention, attenuated, geneposition-shifted RSV are produced in which the recombinant genome orantigenome is further modified by introducing one or more attenuatingmutations specifying an attenuating phenotype in the resultant virus orsubviral particle. These attenuating mutations may be generated de novoand tested for attenuating effects according to a rational designmutagenesis strategy. Alternatively, the attenuating mutations may beidentified in existing biologically derived mutant RSV and thereafterincorporated into a gene position-shifted RSV of the invention.

[0032] In combination with the gene positional changes introduced inrecombinant RSV of the invention, it is often desirable to adjust theattenuation phenotype by introducing additional mutations that increaseor decrease attenuation of the chimeric virus. Thus, candidate vaccinestrains can be further attenuated by incorporation of at least one, andpreferably two or more different attenuating mutations, for examplemutations identified from a panel of known, biologically derived mutantRSV strains. Preferred human mutant RSV strains are cold passaged (cp)and/or temperature sensitive (ts) mutants, for example the mutantsdesignated “cpts RSV 248 (ATCC VR 2450), cpts RSV 248/404 (ATCC VR2454), cpts RSV 248/955 (ATCC VR 2453), cpts RSV 530 (ATCC VR 2452),cpts RSV 530/1009 (ATCC VR 2451), cpts RSV 530/1030 (ATCC VR 2455), RSVB-1 cp52/2B5 (ATCC VR 2542), and RSV B-1 cp-23 (ATCC VR 2579)” (eachdeposited under the terms of the Budapest Treaty with the American TypeCulture Collection (ATCC) of 10801 University Boulevard, Manassas, Va.20110-2209, U.S.A., and granted the above identified accession numbers).From this exemplary panel of biologically derived mutants, a large“menu” of attenuating mutations are provided which can each be combinedwith any other mutation(s) within the panel for calibrating the level ofattenuation in the recombinant, human-bovine chimeric RSV for vaccineuse. Additional mutations may be derived from RSV having non-ts andnon-cp attenuating mutations as identified, e.g., in small plaque (sp),cold-adapted (ca) or host-range restricted (hr) mutant strains. Themutations may be incorporated in either a human or bovine antigenomicsequence, and attenuating mutations identified in a human, bovine orother RSV mutant may be transferred to the heterologous RSV recipient(e.g., bovine or human RSV, respectively) by mapping the mutation to thecorresponding, homologous site in the recipient genome and mutating thenative sequence in the recipient to the mutant genotype (either by anidentical or conservative mutation), as described in U.S. ProvisionalPatent Application No. 60/129,006, filed by Murphy et al. on Apr. 13,1999, incorporated herein by reference.

[0033] Thus, in more detailed embodiments of the invention, geneposition-shifted RSV are provided wherein the recombinant genome orantigenome incorporates at least one and up to a full complement ofattenuating mutations present within a panel of mutant human RSVstrains, said panel comprising cpts RSV 248 (ATCC VR 2450), cpts RSV248/404 (ATCC VR 2454), cpts RSV 248/955 (ATCC VR 2453), cpts RSV 530(ATCC VR 2452), cpts RSV 530/1009 (ATCC VR 2451), cpts RSV 530/1030(ATCC VR 2455), RSV B-1 cp52/2B5 (ATCC VR 2542), and RSV B-1 cp-23 (ATCCVR 2579). In certain embodiments, the recombinant genome or antigenomeincorporates attenuating mutations adopted from different mutant RSVstrains.

[0034] Gene position-shifted RSV designed and selected for vaccine useoften have at least two and sometimes three or more attenuatingmutations to achieve a satisfactory level of attenuation for broadclinical use. In one embodiment, at least one attenuating mutationoccurs in the RSV polymerase gene L (either in the donor or recipientgene) and involves one or more nucleotide substitution(s) specifying anamino acid change in the polymerase protein specifying an attenuationphenotype which may or may not involve a temperature-sensitive (ts)phenotype. Gene position-shifted RSV of the invention may incorporate anattenuating mutation in any additional RSV gene besides L, e.g., in theM2 gene. However, preferred human-bovine chimeric RSV in this contextincorporate one or more nucleotide substitutions in the large polymerasegene L resulting in an amino acid change at amino acid Asn43, Cys319,Phe 521, Gln831, Met1169, Tyr1321 and/or His 1690 in the RSV polymerasegene L, as exemplified by the changes, Ile for Asn43, Leu for Phe521,Leu for Gln831, Val for Met1169, and Asn for Tyr1321. Other alternativeamino acid changes, particularly conservative changes with respect toidentified mutant residues, at these positions can of course be made toyield a similar effect as the identified, mutant substitution.Additional desired mutations for incorporation into human-bovinechimeric RSV of the invention include attenuating mutations specifyingan amino acid substitution at Va1267 in the RSV N gene, Glu218 and/orThr523 in the RSV F gene, and a nucleotide substitution in thegene-start sequence of gene M2. Any combination of one or moreattenuating mutations identified herein, up to and including a fullcomplement of these mutations, may be incorporated in human-bovinechimeric RSV to yield a suitably attenuated recombinant virus for use inselected populations or broad populations of vaccine recipients.

[0035] In other more detailed embodiments of the invention, geneposition-shifted RSV are provided wherein the recombinant genome orantigenome incorporates at least one and up to a full complement ofattenuating mutations specifying an amino acid substitution at Va1267 inthe RSV N gene, Glu218 and/or Thr523 in the RSV F gene, Asn43, Cys319,Phe 521, Gln831, Met1169, Tyr1321 and/or His 1690 in the RSV polymerasegene L, and a nucleotide substitution in the gene-start sequence of geneM2. In certain aspects, the recombinant genome or antigenomeincorporates at least two, commonly three, four or five, and sometimes afull complement comprising all of these attenuating mutations. Often, atleast one attenuating mutation is stabilized by multiple nucleotidechanges in a codon specifying the mutation.

[0036] Attenuating mutations for incorporation in human-bovine chimericRSV of the invention may be selected in coding portions of a donor orrecipient RSV gene or in non-coding regions such as a cis-regulatorysequence. Exemplary non-coding mutations include single or multiple basechanges in a gene start sequence, as exemplified by a single or multiplebase substitution in the M2 gene start sequence at nucleotide 7605(nucleotide 7606 in recombinant sequence).

[0037] Infectious RSV according to the invention can incorporateheterologous, coding or non-coding nucleotide sequences from anyheterologous RSV or RSV-like virus, e.g., human, bovine, murine(pneumonia virus of mice), or avian (turkey rhinotracheitis virus)pneumovirus, or from another enveloped virus, e.g., parainfluenza virus(PIV). Exemplary heterologous sequences include RSV sequences from onehuman RSV strain combined with sequences from a different human RSVstrain, or RSV sequences from a human RSV strain combined with sequencesfrom a bovine RSV strain. Gene position-shifted RSV of the invention mayincorporate sequences from two or more wild-type or mutant RSV strains,for example mutant strains selected from cpts RSV 248, cpts 248/404,cpts 248/955, cpts RSV 530, cpts 530/1009, or cpts 530/1030.Alternatively, chimeric RSV may incorporate sequences from two or more,wild-type or mutant human RSV subgroups, for example a combination ofhuman RSV subgroup A and subgroup B sequences. In yet additionalaspects, one or more human RSV coding or non-coding polynucleotides aresubstituted with a counterpart sequence from a heterologous RSV ornon-RSV virus, alone or in combination with one or more selectedattenuating mutations, e.g., cp and/or ts mutations, to yield novelattenuated vaccine strains.

[0038] Mutations incorporated within gene position-shifted RSV cDNAs,vectors and viral particles of the invention can be introducedindividually or in combination into a full-length RSV cDNA, and thephenotypes of rescued virus containing the introduced mutations can bereadily determined. In exemplary embodiments, amino acid changesdisplayed by attenuated, biologically-derived viruses versus a wild-typeRSV, for example changes exhibited by cpRSV or tsRSV, are incorporatedin combination within a gene position-shifted RSV to yield a desiredlevel of attenuation.

[0039] In additional aspects of the invention, gene position-shifted RSVcan be readily designed as “vectors” to incorporate antigenicdeterminants from different pathogens, including more than one RSVstrain or group (e.g., both human RSV A and RSV B subgroups), humanparainfluenza virus (HPIV) including HPIV3, HPIV2 and HPIV1, measlesvirus and other pathogens (see, e.g., U.S. Provisional PatentApplication Serial No. 60/170,195; U.S. patent application Ser. No.09/458,813; and U.S. patent application Ser. No. 09/459,062, eachincorporated herein by reference). Within various embodiments, therecombinant genome or antigenome comprises a partial or complete RSV“vector genome or antigenome” combined with one or more heterologousgenes or genome segments encoding one or more antigenic determinants ofone or more heterologous pathogens. The heterologous pathogen may be aheterologous RSV (i.e., a RSV of a different strain or subgroup), andthe heterologous gene or genome segment may encode a RSV NS1, NS2, N, P,M, SH, M2(ORF1), M2(ORF2), L, F or G protein or fragment (e.g., aimmunogenic domain or epitope) thereof. For example, the vector genomeor antigenome may be a partial or complete RSV A genome or antigenomeand the heterologous gene(s) or genome segment(s) may encode antigenicdeterminant(s) of a RSV B subgroup virus.

[0040] In alternative embodiments, the gene position-shifted RSV vectorgenome or antigenome is a partial or complete BRSV genome or antigenomeand the heterologous gene(s) or genome segment(s) encoding the antigenicdeterminant(s) is/are of one or more HRSV(s). For example, the partialor complete BRSV genome or antigenome may incorporate one or moregene(s) or genome segment(s) encoding one or more HRSV glycoproteingenes selected from F, G and SH, or one or more genome segment(s)encoding cytoplasmic domain, transmembrane domain, ectodomain orimmunogenic epitope portion(s) of F, G, and/or SH of HRSV.

[0041] As noted above, gene position-shifted RSV designed as vectors forcarrying heterologous antigenic determinants may incorporate one or moreantigenic determinants of a non-RSV pathogen, such as a humanparainfluenza virus (HPIV). In one exemplary embodiment, one or moreHPIV1, HPIV2, or HPIV3 gene(s) or genome segment(s) encoding one or moreHN and/or F glycoprotein(s) or antigenic domain(s), fragment(s) orepitope(s) thereof is/are added to or incorporated within the partial orcomplete HRSV vector genome or antigenome. In more detailed embodiments,a transcription unit comprising an open reading frame (ORF) of an HPIV1,HPIV2, or HPIV3 HN or F gene is added to or incorporated within therecombinant vector genome or antigenome.

[0042] In yet additional alternate embodiments, the vector genome orantigenome comprises a partial or complete HRSV or BRSV genome orantigenome and the heterologous pathogen is selected from measles virus,subgroup A and subgroup B respiratory syncytial viruses, mumps virus,human papilloma viruses, type 1 and type 2 human immunodeficiencyviruses, herpes simplex viruses, cytomegalovirus, rabies virus, EpsteinBarr virus, filoviruses, bunyaviruses, flaviviruses, alphaviruses andinfluenza viruses. Based on this exemplary list of candidate pathogens,the selected heterologous antigenic determinant(s) may be selected frommeasles virus HA and F proteins, subgroup A or subgroup B respiratorysyncytial virus F, G, SH and M2 proteins, mumps virus HN and F proteins,human papilloma virus L1 protein, type 1 or type 2 humanimmunodeficiency virus gp160 protein, herpes simplex virus andcytomegalovirus gB, gC, gD, gE, gG, gH, gI, gJ, gK, gL, and gM proteins,rabies virus G protein, Epstein Barr Virus gp350 protein; filovirus Gprotein, bunyavirus G protein, Flavivirus E and NS1 proteins, andalphavirus E protein, and antigenic domains, fragments and epitopesthereof. In one embodiment, the heterologous pathogen is measles virusand the heterologous antigenic determinant(s) is/are selected from themeasles virus HA and F proteins and antigenic domains, fragments andepitopes thereof. To achieve such a chimeric construct, a transcriptionunit comprising an open reading frame (ORF) of a measles virus HA genemay be added to or incorporated within a HRSV vector genome orantigenome.

[0043] The present invention thus provides gene position-shifted RSVclones, polynucleotide expression constructs (also referred to asvectors) and particles which can incorporate multiple,phenotype-specific mutations introduced in selected combinations intothe gene position-shifted RSV genome or antigenome to produce anattenuated, infectious virus or subviral particle. This process coupledwith routine phenotypic evaluation provides gene position-shifted RSVhaving such desired characteristics as attenuation, temperaturesensitivity, altered immunogenicity, cold-adaptation, small plaque size,host range restriction, etc. Mutations thus identified are compiled intoa “menu” and introduced in various combinations to calibrate a vaccinevirus to a selected level of attenuation, immunogenicity and stability.

[0044] In yet additional aspects of the invention, gene position-shiftedRSV, with or without attenuating mutations, are constructed to have anucleotide modification to yield a desired phenotypic, structural, orfunctional change. Typically, the selected nucleotide modification willspecify a phenotypic change, for example a change in growthcharacteristics, attenuation, temperature-sensitivity, cold-adaptation,plaque size, host range restriction, or immunogenicity. Structuralchanges in this context include introduction or ablation of restrictionsites into RSV encoding cDNAs for ease of manipulation andidentification.

[0045] In certain embodiments, nucleotide changes within geneposition-shifted RSV include modification of a viral gene by deletion ofthe gene or ablation of its expression. Target genes for mutation inthis context include the attachment (G) protein, fusion (F) protein,small hydrophobic (SH), RNA binding protein (N), phosphoprotein (P), thelarge polymerase protein (L), the transcription elongation factor (M2ORF 1), the RNA regulatory factor M2 ORF2, the matrix (M) protein, andtwo nonstructural proteins, NS1 and NS2. Each of these proteins can beselectively deleted, substituted or rearranged, in whole or in part,alone or in combination with other desired modifications, to achievenovel chimeric RSV recombinants.

[0046] In one aspect of the invention, an SH, NS1, NS2, G or M2-2 geneis modified in the gene position-shifted RSV. For example, each of thesegenes may be deleted or its expression ablated (e.g., by introduction ofa stop codon) to alter the phenotype of the resultant recombinant RSVclone to improve growth, attenuation, immunogenicity or other desiredphenotypic characteristics. For example, deletion of the SH gene in therecombinant genome or antigenome will yield a RSV having novelphenotypic characteristics such as enhanced growth in vitro and/orattenuation in vivo. In a related aspect, an SH gene deletion, ordeletion of another selected non-essential gene or genome segment suchas a NS1, NS2, G or M2-2 gene deletion is constructed in a geneposition-shifted RSV, alone or in combination with one or more differentmutations specifying an attenuated phenotype, e.g., a point mutationadopted directly (or in modified form, e.g., by introducing multiplenucleotide changes in a codon specifying the mutation) from abiologically derived attenuated RSV mutant. For example, the SH, NS 1,NS2, G or M2-2 gene may be deleted in combination with one or more cpand/or ts mutations adopted from cpts248/404, cpts530/1009, cpts530/1030or another selected mutant RSV strain, to yield a gene position-shiftedRSV having increased yield of virus, enhanced attenuation, improvedimmunogenicity and genetic resistance to reversion from an attenuatedphenotype due to the combined effects of the different mutations.

[0047] Alternative nucleotide modifications can include a deletion,insertion, addition or rearrangement of a cis-acting regulatory sequencefor a selected gene in the gene position-shifted RSV. In one example, acis-acting regulatory sequence of one RSV gene is changed to correspondto a heterologous regulatory sequence, which may be a counterpartcis-acting regulatory sequence of the same gene in a different RSV or acis-acting regulatory sequence of a different RSV gene. For example, agene end signal may be modified by conversion or substitution to a geneend signal of a different gene in the same RSV strain. In otherembodiments, the nucleotide modification may comprise an insertion,deletion, substitution, or rearrangement of a translational start sitewithin the chimeric genome or antigenome, e.g., to ablate an alternativetranslational start site for a selected form of a protein. In oneexample, the translational start site for a secreted form of the RSV Gprotein is ablated to modify expression of this form of the G proteinand thereby produce desired in vivo effects.

[0048] In addition, a variety of other genetic alterations can beproduced in a gene position-shifted RSV genome or antigenome, alone ortogether with one or more attenuating mutations adopted from abiologically derived mutant RSV. For example, genes or genome segmentsfrom non-RSV sources may be inserted in whole or in part. Nontranslatedgene sequences can be removed, e.g., to increase capacity for insertingforeign sequences. In yet additional aspects, polynucleotide moleculesor vectors encoding the gene position-shifted RSV genome or antigenomecan be modified to encode non-RSV sequences, e.g., a cytokine, aT-helper epitope, a restriction site marker, or a protein of a microbialpathogen (e.g., virus, bacterium or fungus) capable of eliciting aprotective immune response in an intended host. Different or additionalmodifications in the gene position-shifted RSV antigenome can be made tofacilitate manipulations, such as the insertion of unique restrictionsites in various intergenic regions (e.g., a unique StuI site betweenthe G and F genes) or elsewhere.

[0049] All of the foregoing modifications within the geneposition-shifted RSV genome or antigenome, including nucleotideinsertions, rearrangements, deletions or substitutions yielding pointmutations, site-specific nucleotide changes, and changes involvingentire genes or genome segments, may be made to either a partial orcomplete RSV genome or antigenome, or within a heterologous donor geneor genome segment or recipient, background genome or antigenome in achimeric RSV. In each case, these alterations will preferably specifyone or more phenotypic change(s) in the resulting recombinant RSV, suchas a phenotypic change that results in attenuation,temperature-sensitivity, cold-adaptation, small plaque size, host rangerestriction, alteration in gene expression, or a change in animmunogenic epitope.

[0050] In another aspect of the invention, compositions (e.g., isolatedpolynucleotides and vectors incorporating an RSV-encoding cDNA) andmethods are provided for producing an isolated infectious geneposition-shifted RSV. Using these compositions and methods, infectiousgene position-shifted RSV particles or subviral particles are generatedfrom a recombinant RSV genome or antigenome coexpressed with anucleocapsid (N) protein, a nucleocapsid phosphoprotein (P), a large (L)polymerase protein, and an RNA polymerase elongation factor. In relatedaspects of the invention, compositions and methods are provided forintroducing the aforementioned structural and phenotypic changes into arecombinant gene position-shifted RSV to yield infectious, attenuatedvaccine viruses.

[0051] In one embodiment, an expression vector is provided whichcomprises an isolated polynucleotide molecule encoding a geneposition-shifted RSV genome or antigenome. Also provided is the same ordifferent expression vector comprising one or more isolatedpolynucleotide molecules encoding N, P, L and RNA polymerase elongationfactor proteins. These proteins also can be expressed directly from thegenome or antigenome cDNA. The vector(s) is/are preferably expressed orcoexpressed in a cell or cell-free lysate, thereby producing aninfectious gene position-shifted RSV particle or subviral particle.

[0052] The RSV genome or antigenome and the N, P, L and RNA polymeraseelongation factor (preferably the product of the M2(ORF1) of RSV)proteins can be coexpressed by the same or different expression vectors.In some instances the N, P, L and RNA polymerase elongation factorproteins are each encoded on different expression vectors. Thepolynucleotide molecule encoding the gene position-shifted RSV genome orantigenome is operably joined to these control sequences to allowproduction of infectious virus or viral particles therefrom. Inalternative aspects of the invention, the gene position-shifted RSVgenome or antigenome can include sequences from multiple human RSVstrains or subgroups (A and B), as well as other non-human (e.g.,murine) RSV sequences. In other alternate aspects, the geneposition-shifted RSV genome or antigenome can incorporate non-RSVsequences, for example a polynucleotide containing sequences from humanand bovine RSV operably joined with a nucleotide or polynucleotideencoding a point mutation, protein, protein domain or immunogenicepitope of PIV or another negative stranded RNA virus.

[0053] The above methods and compositions for producing geneposition-shifted RSV yield infectious viral or subviral particles, orderivatives thereof. An infectious virus is comparable to the authenticRSV virus particle and is infectious as is. It can directly infect freshcells. An infectious subviral particle typically is a subcomponent ofthe virus particle which can initiate an infection under appropriateconditions. For example, a nucleocapsid containing the genomic orantigenomic RNA and the N, P, L and M2(ORF1) proteins is an example of asubviral particle which can initiate an infection if introduced into thecytoplasm of cells. Subviral particles provided within the inventioninclude viral particles which lack one or more protein(s), proteinsegment(s), or other viral component(s) not essential for infectivity.

[0054] In other embodiments the invention provides a cell or cell-freelysate containing an expression vector which comprises an isolatedpolynucleotide molecule encoding a gene position-shifted RSV genome orantigenome as described above, and an expression vector (the same ordifferent vector) which comprises one or more isolated polynucleotidemolecules encoding the N, P, L and RNA polymerase elongation factorproteins of RSV. One or more of these proteins also can be expressedfrom the genome or antigenome cDNA. Upon expression the genome orantigenome and N, P, L, and RNA polymerase elongation factor proteinscombine to produce an infectious gene position-shifted RSV viral orsubviral particle.

[0055] Attenuated gene position-shifted RSV of the invention is capableof eliciting a protective immune response in an infected human host, yetis sufficiently attenuated so as to not cause unacceptable symptoms ofsevere respiratory disease in the immunized host. The attenuated virusor subviral particle may be present in a cell culture supernatant,isolated from the culture, or partially or completely purified. Thevirus may also be lyophilized, and can be combined with a variety ofother components for storage or delivery to a host, as desired.

[0056] The invention further provides novel vaccines comprising aphysiologically acceptable carrier and/or adjuvant and an isolatedattenuated gene position-shifted RSV. In one embodiment, the vaccine iscomprised of a gene position-shifted RSV having at least one, andpreferably two or more attenuating mutations or other nucleotidemodifications as described above. The vaccine can be formulated in adose of 10⁶ to 10⁶ PFU of attenuated virus. The vaccine may compriseattenuated gene position-shifted RSV that elicits an immune responseagainst a single RSV strain or antigenic subgroup, e.g. A or B, oragainst multiple RSV strains or subgroups. In this regard, geneposition-shifted RSV of the invention can individually elicit amonospecific immune response or a polyspecific immune response againstmultiple RSV strains or subgroups. Gene position-shifted RSV can becombined in vaccine formulations with other RSVs having differentimmunogenic characteristics for more effective protection against one ormultiple RSV strains or subgroups.

[0057] In related aspects, the invention provides a method forstimulating the immune system of an individual to elicit an immuneresponse against one or more strains or subgroups of RSV in a mammaliansubject. The method comprises administering a formulation of animmunologically sufficient amount of an attenuated, geneposition-shifted RSV in a physiologically acceptable carrier and/oradjuvant. In one embodiment, the immunogenic composition is a vaccinecomprised of gene position-shifted RSV having at least one, andpreferably two or more attenuating mutations or other nucleotidemodifications specifying a desired phenotype as described above. Thevaccine can be formulated in a dose of 10³ to 10⁶ PFU of attenuatedvirus. The vaccine may comprise attenuated gene position-shifted RSVvirus that elicits an immune response against a single RSV strain orantigenic subgroup, e.g. A or B, or against multiple RSV strains orsubgroups. In this context, the gene position-shifted RSV can elicit amonospecific immune response or a polyspecific immune response againstmultiple RSV strains or subgroups. Alternatively, gene position-shiftedRSV having different immunogenic characteristics can be combined in avaccine mixture or administered separately in a coordinated treatmentprotocol to elicit more effective protection against one RSV strain, oragainst multiple RSV strains or subgroups. Preferably the immunogeniccomposition is administered to the upper respiratory tract, e.g., byspray, droplet or aerosol. Often, the composition will be administeredto an individual seronegative for antibodies to RSV or possessingtransplacentally acquired maternal antibodies to RSV.

BRIEF DESCRIPTION OF THE DRAWINGS

[0058]FIG. 1. Shift of the G gene, or the F gene, or the G and F genestogether, to a promoter-proximal position within the RSV genome. Thediagram at the top illustrates the RSV genome designated Blp/ΔSH, inwhich the SH gene has been deleted as described previously (Whitehead etal., J. Virol. 73:3438-3442, 1999; incorporated herein by reference) anda BlpI restriction enzyme site has been added to the upstream noncodingregion of the promoter-proximal NS1 gene. The gene-end (GE) signal ofthe M gene has an asterisk to indicate that it contains a singlenucleotide change incorporated during deletion of the SH gene that makesit identical to the naturally-occurring SH GE signal, as describedpreviously (Whitehead et al., J. Virol. 73:3438-3442, 1999; incorporatedherein by reference). The two boxes underneath the diagram show detailsof the structure of genome Blp/ΔSH as well as modifications that weremade to the promoter-proximal end (left hand box) and M-G-F-M2 region(right hand box) of the ΔSH/Blp genome to create genomes G1/ΔSH, F1/ΔSHand G1F2/ΔSH. All manipulations were performed with a cloned cDNA of RSVantigenomic RNA (Collins et al., Proc. Natl. Acad. Sci. USA92:11563-11567, 1995; Collins et al., Virology 259:251-255, 1999; andWhitehead et al., J. Virol. 73:3438-3442, 1999; incorporated herein byreference), and nucleotide positions are numbered according to thecomplete antigenomic sequence of wild type recombinant RSV (containingthe SH gene) (Collins et al., Proc. Natl. Acad. Sci. USA 92:11563-11567,1995; Murphy et al., U.S. Pat. No. 5,993,824; each incorporated hereinby reference). Note that “upstream” and “downstream” refer to thepromoter-proximal and promoter-distal directions, respectively (thepromoter is at the 3′ leader end of negative-sense genomic RNA, which isat the left hand end as drawn in FIG. 1).

[0059] Genome Blp/ΔSH: nucleotides 92 and 97 of the previously-describedSH-deletion mutant of RSV (Whitehead et al., J. Virol. 73:3438-3442,1999; incorporated herein by reference) were changed from G and A(indicated in the top diagram in the left hand box with small caseletters), respectively, to C and C (bold capital letters), therebycreating a BlpI site (underlined) one nucleotide in front of the ATGtranslational start codon (italicized, bold) of the NS1 open readingframe (ORF). The M-G-F region of the Blp/ΔSH genome (right hand box)illustrates the SH deletion (the SH gene normally lies between the M andG genes).

[0060] Genome G1/ΔSH: This genome contains the G gene in thepromoter-proximal position, inserted into the BlpI site (left hand box).The G cDNA insert was constructed as follows: the complete G ORF anddownstream G noncoding sequence and GE signal (nucleotides 4692 to 5596)were engineered to be followed by a 6-nucleotide IG sequence(representing the first 6 nucleotides of the naturally-occurring G-F IGsequence, CATATT (SEQ ID NO: 1) followed by a copy of the 10-nucleotideGS signal of the NS1 gene (boxed). This cloned sequence was flanked byBlpI sites, and was cloned into the BlpI site of genome Blp/ASH. Thisplaced the G ORF, under the control of RSV GS and GE signals, into thepromoter-proximal position. In the same genome, the G gene was deletedfrom its downstream position between the M and F genes (the point ofdeletion is indicated with a large arrow), and the M and F genes werenow separated only by the G-F IG sequence.

[0061] Genome F1/ΔSH: This genome contains the F gene in thepromoter-proximal position, inserted into the BlpI site. The F cDNAinsert was constructed as follows: the complete F ORF and downstreamnoncoding sequence and GE signal (nucleotides 5662 to 7551) wereengineered to be followed by a 6-nucleotide IG sequence (representingthe first 6 nucleotides of the naturally-occurring F-M2 IG sequence,CACAAT (SEQ ID NO: 2) followed by the 10-nucleotide GS signal of the NS1gene. This cloned sequence was flanked by BlpI sites and was cloned intothe BlpI site of genome Blp/ΔSH. This placed the F ORF, under thecontrol of RSV GS and GE signals, into the promoter-proximal position.In the same genome, the F gene was deleted from its downstream positionbetween the G and M2 genes (the point of deletion is indicated with alarge arrow), and these two genes were now separated only by the F-M2 IGsequence.

[0062] Genome G1F2/ΔSH: This genome contains the G and F genes in thefirst and second promoter-proximal locations, respectively, inserted asa single cDNA into the BlpI site. This G-F cDNA insert was constructedto contain (in upstream to downstream order): the complete G ORF, itsdownstream noncoding and GE signal, the G-F IG sequence, the complete Fgene, 6 nucleotides from the F-M2 IG sequence (CACAAT) (SEQ ID NO: 2),and the NS1 GS signal. This cDNA was flanked by BlpI sites and wascloned into the BlpI site of genome Blp/ASH. This placed the G and Fgenes into positions 1 and 2, respectively, relative to the promoter. Inthe same genome, the G and F genes were deleted from their downstreampositions between the M and M2 genes (the point of deletion is indicatedwith a large arrow), and M and M2 genes were now separated only by theF-M2 IG sequence.

[0063]FIG. 2. Production of infectious recombinant virus during thetransfection and initial passages in vitro. HEp-2 cells were transfectedwith the indicated individual antigenomic plasmid and the N, P, L andM2-1 support plasmids as described (Murphy et al., U.S. Pat. No.5,993,824). The medium supernatants were harvested 3 days later andsubjected to serial undiluted passage in HEp-2 cells, with the mediumsupernatant harvested at 3- to 7-day intervals. Samples of each harvestwere taken, flash-frozen, and analyzed later in parallel by plaqueassay. The viruses were given the same designations as their respectivecDNAs, i.e. Blp/ΔSH, G1/ΔSH etc. Data are shown for Blp/ΔSH, G1/ΔSH,F1/ΔSH and G1F2/ΔSH. Transfection was at 32° C. and subsequent passageswere at 37° C.

[0064]FIG. 3A Replication in vitro of recombinant wt RSV (containing theSH gene), and the ΔSH (not containing a BlpI site), Blp/ΔSH, G1/ΔSH andG1F2/ΔSH mutant viruses following infection at an input multiplicity ofinfection (MOI) of 0.1. Replicate cultures of Vero (top panel) or HEp-2cells (lower panel) were infected and incubated at 37° C. At theindicated time points, duplicate monolayers were harvested for eachvirus and the medium supernatants were flash-frozen. These were analyzedlater in parallel by plaque assay.

[0065]FIG. 3B depicts single-step growth of the Blp/SH, G1/SH, F1/SH andG1F2/SH viruses following infection of Hep-2 (top) and Vero (bottom)cell monolayers at an input multiplicity of infection of 3.0. Replicatecell monolayers were infected and incubated at 37° C., and at theindicated time points duplicate monolayers were harvested and the mediumsupernatants were flash-frozen.

[0066]FIG. 4. Western blot analysis of the expression of the G proteinby Blp/ΔSH, G1/ΔSH and G1F2/ΔSH viruses in Vero cells. The cells fromeach time point in the top panel of FIG. 3A were harvested and the totalprotein was analyzed by gel electrophoresis and Western blotting. Theblots were developed by incubation with a rabbit antiserum specific topeptide of the G protein. Bound antibodies were then visualized bychemiluminescence. The G protein migrates as two forms: the 90 kDamature form and a 50 kDa incompletely-glycosylated form.

[0067]FIG. 5. Structures of attenuated RSV's in which the G and F geneshave been shifted to positions 1 and 2. Panel A: Structure ofrecombinant RSV G1F2/ΔNS2ΔSH, in which the SH and NS2 genes were deletedas described (Teng and Collins, J. Virol. 73:466-473, 1999; Whitehead etal., J. Virol. 73:3438-3442, 1999; incorporated herein by reference),and the G and F genes were moved into positions 1 and 2, respectively,as described above in FIG. 1. Panel B: Structure of recombinant RSVG1F2/ΔNS1ΔNSΔSH, in which the SH, NS1 and NS2 genes were deleted and theG and F genes were moved into positions 1 and 2, respectively.

[0068]FIG. 6 details construction of a chimeric rBRSV/HRSV genome inwhich the BRSV G and F genes have been deleted and the G and F genes ofHRSV have been placed in a promoter-proximal position. BRSV genes areshaded; HRSV genes are clear. Nucleotide sequence position numbers arerelative to the complete rBRSV antigenome (Buchholz et al., J. Virol.73:251-259, 1999; Buchholz et al., J. Virol. 74:1187-1199, 2000; GenBankaccession number AF092942 or complete rHRSV antigenome in Collins etal., Proc. Natl. Acad. Sci. USA 92:11563-11567, 1995; each incorporatedherein by reference); sequence position numbers that refer to the HRSVsequence are underlined. FIG. 6, panel A details structure of rBRSVcontaining NotI, SalI and XhoI sites that were added in previous work(Buchholz et al., J. Virol. 73:251-259, 1999; Buchholz et al., J. Virol.74:1187-1199, 2000). FIG. 6, panel B depicts modifications to rBRSV tocreate rBRSV/A2-G1F2. The BRSV G and F genes were deleted by digestionwith SalI and XhoI and religation of the resulting compatible cohesiveends. The region of the genome of HRSV from nucleotides 4692 to 7557,containing the G and F genes, was amplified by PCR using primers thatcontained desired changes to be incorporated into each end of the cDNA.The amplified PCR product contained (in upstream to downstream order): aNotI site, a BlpI site, the complete HRSV G ORF, its downstreamnoncoding and GE signal, the HRSV G-F IG sequence, the complete HRSV Fgene, 6 nucleotides from the HRSV F-M2 IG sequence (CACAAT), the NS1 GSsignal, a BlpI site and a NotI site. This cDNA was cloned into theunique Notl site at position 67 of rBRSV. FIG. 6, panel C illustratesstructure of the genomic RNA of rBRSV/A2-G1F2.

[0069]FIG. 7 depicts multicycle growth of RBRSV, rHRSV(rA2), rBRSV/A2,and rBRSV/A2-G1 F2 in HEp-2 human (left panel) and MDBK bovine (rightpanel) cells. Duplicate cell monolayers were infected with the indicatedvirus at an MOI of 0.1 and incubated at 37° C., and medium aliquots wereharvested at the indicated times, flash frozen, stored at −70° C. andtitrated later in duplicate. Each value is the mean titer of two wells.

[0070]FIG. 8 shows indirect immunofluorescence of HEp-2 cells infectedwith rBRSV/A2-G1F2, rBRSV/A2, or rA2. Cells were infected at an MOI of0.1, incubated at 37° C. for 96 hours, fixed with acetone, permeabilizedand reacted with monoclonal antibody 021/1G, specific to the G proteinof HRSV, or with monoclonal antibody 44F, specific to the F protein ofHRSV. Antibody binding was visualized by reaction with a tagged antibodyspecific to murine IgG.

[0071]FIG. 9 details construction of a chimeric rBRSV/HRSV containingthe M, G and F genes of HRSV. BRSV genes are shaded; HRSV genes areclear. Sequence position numbers that refer to HRSV genes areunderlined. FIG. 9, panel A depicts modification of rBRSV/A2 to containa unique MluI site at position 3204, within the intergenic regionbetween the P and M genes (P-M IG). The sequence of the IG is shown,with small case letters indicating the original nucleotide assignments.The underlined letters indicate the Mlul site created by the 5nucleotide substitutions. Nucleotide sequence position numbers arerelative to the complete rBRSV antigenome (Buchholz et al., J. Virol.73:251-259, 1999; Buchholz et al., J. Virol. 74:1187-1199, 2000; GenBankaccession number AF092942 or complete rHRSV antigenome in Collins etal., Proc. Natl. Acad. Sci. USA 92:11563-11567, 1995); sequence positionnumbers that refer to the HRSV sequence are underlined. FIG. 9, panel Billustrates modification of rBRSV/A2 to create rBRSV/A2-MGF. TheMluI-SalI fragment containing the BRSV M and SH genes was excised andreplaced with an MluI-SalI fragment containing the HRSV M gene. TheMluI-SalI fragment containing the HRSV M gene is shown in the box. Alsoshown is the sequence immediately upstream of the MluI site, includingthe BRSV P gene-end sequence, and the sequence immediately downstream ofthe SalI site, including the intergenic sequence between the M and Ggenes (M-G IG), the HRSV G gene-start signal, and the ATG (bold,italics) that begins the G ORF. FIG., 9, panel C depicts the structureof the genome of rBRSV/A2-MGF.

[0072]FIG. 10 depicts the structures of the genomes of recombinant BRSV(rBRSV, top) and five BRSV/HRSV chimeric viruses in which specific BRSVgenes (shaded rectangles) were replaced with their HRSV counterparts(open rectangles). In addition, in the bottom two viruses the G and Fgenes were moved from their normal positions to positions 3 and 4 or 1and 2. In the diagram of rBRSV, several restriction sites are indicated.Restriction sites used in the various constructions are indicated: theKpnl site occurs naturally and the others were introduced as necessary(Buchholz, et al., J. Virol., 73:251-9, 1999; Buchholz, et al., J.Virol., 74:1187-1199, 2000, each incorporated herein by reference).

[0073]FIG. 11 depicts multicycle growth of the BRSV/HRSV chimericviruses rBRSV/A2-G3F4 (top panel) and HEx (bottom panel) compared torHRSV (rA2) and rBRSV parental viruses as well as thepreviously-described chimeric viruses rBRSV/A2-GF (previously calledrBRSV/A2; Buchholz, 2000, supra) and rBRSV/A2-G1F2. Monolayer culturesof Vero cells were infected at an input multiplicity of infection of 0.1and incubated at 37° C. Samples from the overlying medium were harvestedat the indicated times and virus titers were determined by the limitingdilution method. To 0.1 ml of serial 10-fold virus dilutions per well,104 BHK-21 cells were added in a 0.1 ml volume. After 48 hours, cellswere fixed in 80% acetone, and an indirect immunofluorescence assayusing an antibody specific to the BRSV M protein, cross-reactive withthe HRSV M protein, was done, and foci of infected cells were counted(see, Buchholz et al., J. Virol. 73:251-9, 1999 (incorporated herein byreference).

[0074]FIG. 12 compares the size of the plaque or focus formed by theindicated viruses on human HEp-2 cells (top panel) versus bovine MDBKcells (bottom panel), expressed as a percentage compared to HRSV (HEp-2cells) or BRSV (MDBK cells).

[0075]FIG. 13A and 13B depict the structures of the genomes ofrecombinant BRSV in which the N and/or P genes were replaced by theirHRSV counterparts. FIG. 13A shows chimeras in which these substitutionswere made in the rBRSV backbone, and FIG. 13B shows chimeras in whichthe backbone was the rBRSV/A2-NS1+2 virus.

[0076]FIG. 14 Diagram of the genomic RNA of the recombinant rRSV/6120virus containing a deletion in the SH gene, drawn as the negative senseRNA, 3′ to 5′, with each encoded mRNA indicated with a rectangle and nonmRNA-coding extragenic and intergenic regions as a horizontal line. Theparent RSV antigenomic cDNA was as described previously (Collins, et al,Proc. Natl. Acad. Sci. USA 92:11563-11567, 1995), with the furthermodification of an XmaI site in the G-F intergenic region (Bukreyev, etal., J. Virol. 70:6634-6641, 1996, incorporated herein by reference).This cDNA was modified (i) to contain five translationally-silentnucleotide substitutions in the last four codons of the SH ORF includingthe translational stop codon and (ii) to delete 112 nucleotides(positions 4499-4610) of the complete antigenomic sequence from thedownstream non-translated region of the SH gene (box). The XhoI and PacIsites used in the construction are italicized and labeled, the SHgene-end signal is underlined, the SH codons are shown as triplets,nucleotide substitutions are in small case, and the deleted sequence isrepresented with a box with the sequence positions indicated.

[0077] FIGS. 15A-15B Growth kinetics of rRSV/6120, containing a deletionin the SH gene, compared to its full length recombinant rRSV parent D53.Three sets (FIG. 15A, FIG. 15B and FIG. 15C) of monolayer cultures ofHEp-2 cells were infected with the indicated virus at an inputmultiplicity of infection of 0.005. Following an adsorption period,cells were incubated at 37° C. At 12 h intervals, the medium washarvested in its entirety and aliquots were flash-frozen for latertitration. The cells were washed three times and fresh medium was addedand the incubation continued. At the end of the experiment, the sampleswere analyzed by plaque assay to determine virus titer.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

[0078] The present invention provides recombinant respiratory syncytialviruses (RSVs) which are modified by shifting a gene order or spatialposition of one or more genes within a recombinant RSV genome orantigenome to generate a vaccine virus that is infectious and attenuatedin humans and other mammals. Typically, the recombinant RSV genome orantigenome is modified by repositioning one or more “shifted” genes orgene segments, directly or indirectly via introduction, deletion orrearrangement of a second, “displacement polynucleotide” within thegenome, resulting in a positional shift of the subject “shifted” gene orgenome segment to a more promoter-proximal or promoter-distal position.Shifting of gene or genome segment positions in this context isdetermined to a position of the subject gene or genome segment in aparent RSV genome or antigenome prior to introduction of the gene shift,for example relative to the position of the subject gene or genomesegment in a wild type RSV genome or antigenome (e.g., HRSV A2 or BRSVkansas strains) or in a parental recombinant RSV genome or antigenome asdisclosed herein, prior to the gene shift.

[0079] In certain aspects of the invention, the gene position-shiftedRSV features one or more shifted genes or genome segments that areshifted to a more promoter-proximal or promoter-distal position byinsertion, deletion, or rearrangement of one or more displacementpolynucleotides within the partial or complete recombinant RSV genome orantigenome. The displacement polynucleotides may comprise an RSV gene orgenome segment, including an RSV gene or genome segment from a differentor “heterologous” RSV (e.g., in the case of a heterologous gene orgenome segment inserted into genome or antigenome of a different RSV).Alternatively, the displacement polynucleotides may be from a non-RSVsource, including from a non-RSV pathogen such as parainfluenza virus(PIV) or measles virus. The displacement polynucleotides may encode aprotein or a portion of a protein, such an immunogenic domain or epitopeof a glycoprotein, or they may represent an incomplete or impairedcoding sequence, including non-coding and nonsense polynucleotidesequences.

[0080] In certain aspects of the invention, the recombinant RSV featuresone or more positionally shifted genes or genome segments that may beshifted to a more promoter-proximal or promoter-distal position byinsertion, deletion, or rearrangement of one or more displacementpolynucleotides within the partial or complete recombinant RSV genome orantigenome. In certain aspects, the displacement polynucleotides are RSVgenes or genome segments. In other aspects, displacement polynucleotideslack a complete open reading frame (ORF). Within more detailedembodiments, displacement polynucleotides comprise polynucleotideinserts of between 150 nucleotides (nts) and 4,000 nucleotides inlength. Displacement polynucleotides may be inserted or rearranged intoa non-coding region (NCR) of the recombinant genome or antigenome, ormay be incorporated in the recombinant RSV genome or antigenome as aseparate gene unit (GU).

[0081] Gene position shifts within the recombinant RSV of the inventionare typically determined relative to the genome or antigenome“promoter”. The RSV promotor contains the polymerase initiation site,which is a conserved sequence element recognized by the polymerase. Thepromoter is located at the 3′ end of the genome or antigenome, withinapproximately the thirty 3′-terminal nucleotides. In the case of the RSVgenome, the promoter directs both transcription and replication.However, the antigenome “promoter” which lacks transcription signals andonly naturally controls replication can be modified to directtranscription by insertion of known transcription signals. For thepurposes of describing the invention, the RSV promotor is thus construedto reside at the 3′ end of either the genome or antigenome, whereby theterms “promoter-proximal” and “promoter-distal” used herein alternatelyrefer to a direction toward, or away from, respectively, the 3′ end ofthe genome or antigenome.

[0082] Thus provided within the invention are isolated polynucleotidemolecules, vectors (expression constructs), and recombinant virusesincorporating a recombinant RSV genome or antigenome-wherein one or moregenes or gene segments is/are shifted to a more promoter-proximal orpromoter-distal position within the recombinant genome or antigenomecompared to a parental or wild type position of the gene in the RSV genemap. Shifting the position of genes in this manner provides for aselected increase or decrease in expression of one or more positionally“shifted” genes, depending on the nature and degree of the positionalshift. In one embodiment, RSV glycoproteins are upregulated by shiftingone or more glycoprotein-encoding genes to a more promoter-proximalposition. Genes of interest for manipulation to create geneposition-shifted RSV include any of the NS1, NS2, N, P, M, SH, M2(ORF1),M2(ORF2), L, F or G genes or a genome segment that may be part of a geneor extragenic. A variety of additional mutations and nucleotidemodifications are provided within the gene position-shifted RSV of theinvention to yield desired phenotypic and structural effects.

[0083] The recombinant construction of human-bovine RSV yields a viralparticle or subviral particle that is infectious in mammals,particularly humans, and useful for generating immunogenic compositionsfor clinical use. Also provided within the invention are novel methodsand compositions for designing and producing attenuated, geneposition-shifted RSV, as well as methods and compositions for theprophylaxis and treatment of RSV infection. Gene position-shifted RSVand immunogenic compositions according to the invention may elicit animmune response to a specific RSV subgroup or strain, or they may elicita polyspecific response against multiple RSV subgroups or strains. Geneposition-shifted RSV of the invention are thus infectious and attenuatedin humans and other mammals. In related aspects, the invention providesnovel methods for designing and producing attenuated, geneposition-shifted RSV that are useful in various compositions to generatea desired immune response against RSV in a host susceptible to RSVinfection. Included within these aspects of the invention are novel,isolated polynucleotide molecules, vectors, and infected cellsincorporating such molecules that comprise a gene position-shifted RSVgenome or antigenome. Gene position-shifted RSV according to theinvention may elicit an immune response to a specific RSV subgroup orstrain, or a polyspecific response against multiple RSV subgroups orstrains. Yet additional compositions and methods are provided fordesigning and producing attenuated, gene position-shifted RSV as vectorsfor incorporating antigenic determinants of other pathogens to generatea desired immune response against different pathogens of interest. Alsoprovided within the invention are methods and compositions incorporatinggene position-shifted RSV for prophylaxis and treatment of infection anddisease caused by RSV and other pathogens.

[0084] The present invention culminates and supplements a continuingline of discovery founded upon the recent advent and refinement ofmethods for producing infectious recombinant RSV from cDNA. Based uponthis work, it has been possible to directly investigate the roles of RNAand protein structures in RSV gene expression and replication. Theseinvestigations are described or reported in U.S. Provisional PatentApplication No. 60/007,083, filed Sep. 27, 1995; U.S. patent applicationSer. No. 08/720,132, filed Sep. 27, 1996; U.S. Provisional PatentApplication No. 60/021,773, filed Jul. 15, 1996; U.S. Provisional PatentApplication No. 60/046,141, filed May 9, 1997; U.S. Provisional PatentApplication No. 60/047,634, filed May 23, 1997; U.S. Pat. No. 5,993,824,issued Nov. 30, 1999 (corresponding to International Publication No. WO98/02530); U.S. patent application Ser. No. 09/291,894, filed by Collinset al. on Apr. 13, 1999; U.S. Provisional Patent Application No.60/129,006, filed by Murphy et al. on Apr. 13, 1999; Crowe et al.,Vaccine 12: 691-699, 1994; and Crowe et al., Vaccine 12: 783-790, 1994;Collins, et al., Proc Nat. Acad. Sci. USA 92:11563-11567, 1995;Bukreyev, et al., J Virol 70:6634-41, 1996, Juhasz et al., J. Virol.71(8):5814-5819, 1997; Durbin et al., Virology 235:323-332, 1997; Karronet al., J. Infect. Dis. 176:1428-1436, 1997; He et al. Virology237:249-260, 1997; Baron et al. J. Virol. 71:1265-1271, 1997; Whiteheadet al., Virology 247(2):232-9, 1998a; Whitehead et al., J. Virol.72(5):4467-4471, 1998b; Jin et al. Virology 251:206-214, 1998; Bukreyev,et al., Proc. Nat. Acad. Sci. USA 96:2367-2372, 1999; Bermingham andCollins, Proc. Natl. Acad. Sci. USA 96:11259-11264, 1999 Juhasz et al.,Vaccine 17:1416-1424, 1999; Juhasz et al., J. Virol. 73:5176-5180, 1999;Teng and Collins, J. Virol. 73:466-473, 1999; Whitehead et al., J.Virol. 73:9773-9780, 1999; Whitehead et al., J. Virol. 73:871-877, 1999;and Whitehead et al., J. Virol. 73:3438-3442, 1999, Jin, et al.,Virology, 273:210-8, 2000; Jin, et al., J. Virol., 74:74-82, 2000; Teng,et al., J. Virol. 74:9317-21, 2000, each of which are incorporatedherein by reference in their entirety for all purposes).

[0085] With regard to gene position-shifted RSV of the invention, anumber of the foregoing incorporated disclosures have focused onmodification of the naturally-occurring gene order in RSV. For example,each of the NS1, NS2, SH and G genes have been successfully deletedindividually in infectious RSV recombinants, thereby shifting theposition of downstream genes relative to the viral promoter. In otherrecombinants within the invention, the NS 1 and NS2 gene were deletedtogether, shifting the remaining genes in promoter-proximal directionwithin the recombinant RSV genome or antigenome. For example, when NS 1and NS2 are deleted together, N is moved from position 3 to position 1,P from position 4 to position 2, and so on. Deletion of any other RSVgene within alternate embodiments of the invention will similarly shiftthe position (relative to the promoter) of those genes which are locatedfurther downstream. For example, SH occupies position 6 in wild typevirus, and its deletion does not affect M at position 5 (or any otherupstream gene) but moves G from position 7 to 6 relative to thepromoter. It should be noted that gene deletion also can occur (rarely)in a biologically-derived mutant virus (Karron et al., Proc. Natl. Acad.Sci. USA 94:13961-13966, 1997; incorporated herein by reference). Notethat “upstream” and “downstream” refer to the promoter-proximal andpromoter-distal directions, respectively (the promoter is at the 3′leader end of negative-sense genomic RNA).

[0086] Gene order shifting modifications (i.e., positional modificationsmoving one or more genes to a more promoter-proximal or promoter-distallocation in the recombinant viral genome) with gene position-shifted RSVof the invention result in viruses with altered biological properties.For example, RSV lacking NS1, NS2, SH, G, NS1 and NS2 together, or SHand G together, have been shown to be attenuated in vitro, in vivo, orboth. It is likely that this phenotype was due primarily to the loss ofexpression of the specific viral protein. However, the altered gene mapalso likely contributed to the observed phenotype. This effect iswell-illustrated by the SH-deletion virus, which grew more efficientlythan wild type in some cell types, probably due to an increase in theefficiency of transcription, replication or both resulting from the genedeletion and resulting change in gene order and possibly genome size. Inother viruses, such as RSV in which NS1 and/or NS2 were deleted, alteredgrowth that might have occurred due to the change in gene order likelywas obscured by the more dominant phenotype due to the loss ofexpression of the RSV protein(s).

[0087] Yet additional changes have been successfully introduced tochange the gene order of RSV to improve its properties as alive-attenuated vaccine. In specific examples demonstrating efficacy ofthe invention, the RSV G and F genes were shifted, singly and in tandem,to a more promoter-proximal position relative to their wild-type geneorder. These two proteins normally occupy positions 7 (G) and 8 (F) inthe RSV gene order (NS 1 -NS2-N-P-M-SH-G-F-M2-L). In order to increasethe possibility of successful recovery, these positional manipulationsof G and F were performed in a version of RSV in which the SH gene hadbeen deleted (see, e.g., Whitehead et al., J. Virol., 73:3438-42 (1999),incorporated herein by reference). This facilitates viral recoverybecause this virus makes larger plaques in vitro (Bukreyev et al., J.Virol. 71:8973-82, 1997, incorporated herein by reference). G and F werethen moved individually to position 1, or were moved together topositions 1 and 2, respectively.

[0088] Surprisingly, recombinant RSV were readily recovered in which Gor F were moved to position 1, or in which G and F were moved topositions 1 and 2, respectively. This result differed greatly fromprevious reported studies with vesicular stomatitis virus (VSV), wheremovement of the single VSV glycoprotein gene by only two positions wasvery deleterious to virus growth (Ball et al., J. Virol. 73:4705-4712,1999, incorporated herein by reference). The ability to recover thesealtered viruses also was surprising because RSV replicates inefficientlyand because RSV has a complex gene order and movement of theglycoprotein genes involved a large number of position changes. Indeed,the rearranged RSV's grew at least as well as their immediate parenthaving the wild type order of genes. As indicated above, this isparticularly important for RSV, since the wild type virus growsinefficiently in cell culture and a further reduction in replication invitro would likely render vaccine preparation unfeasible. Thus, it isremarkable that all of the NS1-NS2-N-P-M proteins could be displaced byone or two positions relative to the promoter without a significantdecrease in growth fitness. In addition, examination of the expressionof the G glycoprotein showed that it was increased up to several-foldover that of its parent virus. This indicated that a vaccine viruscontaining G and/or F in the first position expresses a higher molaramount of these protective antigens compared to the other viralproteins, and thus represent a virus with desired vaccine properties.

[0089] Similarly extensive modifications in gene order also wereachieved with two highly attenuated RSV vaccine candidates in which theNS2 gene was deleted on its own, or in which the NS1 and NS2 genes weredeleted together, as described in more detail in the above-incorporatedreferences. In these two vaccine candidates, the G and F glycoproteinswere moved together to positions 1 and 2 respectively, and the G, F andSH glycoproteins were deleted from their original downstream position.Thus, the recovered viruses G1F2ΔNS2ΔSH and G1F2/ΔNS1ΔNS2ΔSH had two andthree genes deleted respectively in addition to the shift of the G and Fgenes. To illustrate the extent of the changes involved, the gene ordersof wild type RSV (NS1-NS2-N-P-M-SH-G-F-M2-L) and the G1F2/ΔNS2ΔSH virus(G-F-NS1-N-P-M-M2-L) or the ΔNS1ΔNS2ΔSH (G-F-N-P-M-M2-L) can becompared. This shows that the positions of most or all of the genesrelative to the promoter were changed. Nonetheless, these highlyattenuated derivatives retained the capacity to be grown in cellculture.

[0090] Yet additional changes have been successfully introduced tochange the gene order of RSV in a human-bovine chimeric RSV to improveits properties as a live-attenuated vaccine (See, U.S. patentapplication Ser. No. 09/602,212, filed by Bucholz et al. on Jun. 23,2000, its corresponding PCT application published as WO 01/04335 on Jan.18, 2001, and its priority provisional U.S. application Ser. No.60/143,132 filed on Jul. 9, 1999, each incorporated herein byreference). As illustrated in the examples below, an infectiousrecombinant human-bovine chimeric RSV (rBRSV/HRSV) was successfullyconstructed and recovered in which the HRSV G and F genes aresubstituted into a recombinant bovine RSV (rBRSV) background. Theresulting human-bovine chimera contains two genes of HRSV, namely G andF, and eight genes from BRSV, namely NS1, NS2, N, P, M, SH, M2 and L. Inaddition to this basic substituted glycoprotein construction, the HRSV Gand F genes are shifted to a more promoter-proximal position in therBRSV backbone, i.e., relative to the wild-type gene order position ofthe F and G genes in the RSV genome. More specifically, the F and Ggenes were moved from their usual location relative to the promoter,namely gene positions 7 and 8, respectively, to positions 1 and 2,respectively. The resulting chimeric recombinant virus, rBRSV/A2-G1F2,is very similar in its levels of F and G protein expression as detectedby immunofluorescence to that of wt HRSV, which result is interpreted toshow increased expression of the G and F glycoproteins attributed to thepromoter-proximal shift of the genes. Since the present rBRSV/A2-G1F2virus bears the same constellation of BRSV genes in its geneticbackground, it is likely to share this strong host range restrictionphenotype. In this context, the increased expression of the twoprotective antigens in vivo will increase the immunogenicity of thisvirus to produce highly desirable vaccine properties.

[0091] RSV is a nonsegmented negative strand RNA virus of OrderMononegavirales. The mononegaviruses constitute a large and diverseOrder that includes four families: Family Rhabdoviridae, represented byvesicular stomatitis virus (VSV) and rabies virus; Family Bornaviridae,represented by boma disease virus; Family Filoviridae, represented byMarburg and Ebola viruses, and Family Paramyxoviridae. This latterfamily is further divided into two subfamilies: Paramyxovirinae, whichincludes Sendai, measles, mumps and parainfluenza viruses, andPneumovirinae, which includes respiratory syncytial virus.

[0092] The genome of a mononegavirus is a single strand of RNA thatcontains from 5 (VSV) to 11 (RSV) genes arranged in a linear array. Themononegavirus genome does not encode protein directly (hence thedesignation “negative sense”), but rather encodes complementarypositive-sense mRNAs that each encode one or more proteins. Typically, agene begins with a short gene-start signal and ends with a shortgene-end signal. These signals usually consist of 8 to 12 nucleotidesand usually are highly conserved between genes of a given virus and to alesser extent between related viruses.

[0093] In the case of RSV, the genome is more than 15.2 kb in length andis transcribed into 10 separate major mRNAs that encode 11 identifiedproteins. Specifically, the RSV gene order is3′-NS1-NS2-N-P-M-SH-G-F-M2-L-5′, and the M2 mRNA encodes two proteins,M2-1 and M2-2 from overlapping ORFs. The gene-start and gene-end signalsof RSV, together with sequences involved in RNA replication and inpromoter function, also have been identified and analyzed in ongoingwork (Bukreyev et al., J. Virol. 70:6634-6641, 1996; Collins et al.,Proc. Natl. Acad. Sci. USA 88:9663-9667, 1991; Mink et al., Virology185:615-624, 1991; Grosfeld et al., J. Virol. 69:5677-5686, 1995; Hardyand Wertz, J. Virol. 72:520-526, 1998; Hardy et al., J. Virol.73:170-176, 1999; Kuo et al., J. Virol. 70:6143-6150, 1996; Kuo et al.,J. Virol. 70:6892-6901, 1996; Samal and Collins, J. Virol. 70:5075-5082,1996; Kuo et al., J. Virol. 71:4944-4953, 1997; Feams and Collins, J.Virol. 73:388-397, 1999; each incorporated herein by reference).

[0094] The 3′ end of a mononegavirus genome contains a promoter thatdirects entry of the polymerase (Lamb and Kolakofsky, Fields Virology,1:1177-1204, 1996; and Wagner and Rose, Fields Virology, 1121-1136,1996; each incorporated herein by reference). This promoter is containedcompletely or in part in an extragenic leader region at the 3′ end ofthe genome. The polymerase then transcribes the genome 3′-to-5′ in alinear, stop-restart manner guided by the gene-start and gene-endsignals. The gene-start signal of each gene directs initiation of thesynthesis of the corresponding mRNA and the gene-end signal directspolyadenylation, termination and release of the corresponding mRNA. Thepolymerase then remains template-bound and reinitiates at the nextdownstream gene-start signal. This process is repeated to transcribe onegene after another in their 3′-to-5′ order (Lamb and Kolakofsky, FieldsVirology 1:1177-1204, 1996; Wagner and Rose, Fields Virology, 1121-1136,1996; Abraham and Banerjee, Proc. Natl. Acad. Sci. USA 73:1504-1508,1976; Ball and White, Proc. Natl. Acad. Sci. USA, 73:442-446, 1976;Ball, J. Virol. 21:411-414, 1977; Banerjee et al., J. Gen. Virol.34:1-8, 1977; Iverson and Rose, Cell 23:477-484, 1981; Iverson and Rose,J. Virol. 44:356-365, 1982; Banerjee et al., Pharmacol. Ther. 51:47-70,1991; each incorporated herein by reference).

[0095] Four of the RSV proteins enumerated above arenucleocapsidlpolymerase proteins, namely the major nucleocapsid Nprotein, the phosphoprotein P, and polymerase protein L, and thetranscription antitermination protein M2-1. Three are surfaceglycoproteins, namely the attachment G protein, the fusion Fglycoprotein responsible for penetration and syncytium formation, andthe small hydrophobic SH protein of unknown function. The matrix Mprotein is an internal virion protein involved in virion formation.There are two nonstructural proteins NS1 and NS2 of unknown function.Finally, there is a second open reading frame (ORF) in the M2 mRNA whichencodes an RNA regulatory factor M2-2.

[0096] The G and F proteins are the major neutralization and protectiveantigens (Collins, et al., Fields Virology 2:1313-1352, 1996; Connors,et al., J. Virol. 66:1277-81, 1992). Resistance to reinfection by RSV islargely mediated by serum and mucosal antibodies specific against theseproteins. RSV-specific cytotoxic T cells are also induced by RSVinfection and can be directed against a number of different proteins,but this effector has not yet been shown to be an important contributorto long term resistance to reinfection. However, both CD8+ and CD4+cells can be important in regulating the immune response, and both maybe involved in viral pathogenesis (Johnson, et al., J. Virol.72:2871-80, 1998; Srikiatkhachorn and Braciale, J. Exp. Med. 186:421-32,1997). Thus, F and G are the most important antigenic determinants, butother proteins can also play important roles in the immune response.

[0097] RSV isolates can be segregated into two antigenic subgroups, Aand B, by reactivity with monoclonal antibodies (Anderson, et al., J.Infect. Dis. 151:626-33, 1985, Mufson, et al., J. Gen. Virol.66:2111-24, 1985). The two subgroups exhibit differences across thegenome, but are the most divergent in the ectodomain of the G proteinwhere the percent amino acid sequence divergence can exceed 50% and theantigenic divergence is 95% based on reactivity of monospecificpolyclonal antisera (Johnson, et al., Proc. Natl. Acad. Sci. USA84:5625-9, 1987; Johnson, et al., J. Virol. 61:3163-6, 1987). The Fprotein is approximately 10% divergent by amino acid sequence and 50%divergent antigenically between RSV A and B subgroups (Johnson, et al.,J. Virol. 61:3163-6, 1987; Johnson and Collins, J. Gen. Virol.69:2623-8, 1988). Thus, both subgroups should be represented in avaccine.

[0098] RSV and other mononegaviruses have been reported to exhibit agradient of decreasing gene transcription, such that the mostpromoter-proximal gene is transcribed the most efficiently, and eachgene thereafter displays an incrementally-decreasing efficiency oftranscription (Lamb and Kolakofsky, Fields Virology, 1:1177-1204, 1996;Wagner and Rose, Fields Virology, 1121-1136, 1996; Abraham and Banerjee,Proc. Natl. Acad. Sci. USA 73:1504-1508, 1976; Ball and White, Proc.Natl. Acad. Sci. USA 73:442-446, 1976; Ball, J. Virol. 21:411-414, 1977;Banerjee et al., J. Gen. Virol., 34:1-8, 1977; Iverson and Rose, Cell23:477-484, 1981; Iverson and Rose, J. Virol. 44:356-365, 1982; Banerjeeet al., Pharmacol. Ther. 51:47-70, 1991; each incorporated herein byreference). This gradient of gene expression has been reported andpartially characterized for RSV (Collins and Wertz, Proc. Natl. Acad.Sci. USA 80:3208-3212, 1983; Collins et al., J. Virol. 49:572-578, 1984;Dickens et al., J. Virol. 52:364-369, 1984; each incorporated herein byreference). This gradient is thought to be partially attributed to“fall-off” of the polymerase during sequential transcription. Studieswith the rhabdovirus VSV, one of the simplest of the mononegaviruses,suggest that fall-off occurs primarily at the intergenic regions(Iverson and Rose, Cell 23:477-484, 1981; Iverson and Rose, J. Virol.44:356-365, 1982; each incorporated herein by reference), although thedisproportionately low abundance of the large L mRNA suggests that therealso is significant fall-off within genes.

[0099] The gradient of gene transcription reported among mononegavirusesis thought to be a major factor that determines the relative molarratios of the various viral mRNAs within an infected cell. Thisphenomenon in turn is thought to be a major factor determining therelative molar ratios of viral proteins. All mononegaviruses have genesencoding the following five proteins or counterparts thereof: anRNA-binding nucleocapsid protein N, a phosphoprotein P, an internalvirion matrix protein M, an attachment protein G, HA, or HN, and a largepolymerase protein L. Furthermore, these are always found in the3′-to-5′ order N-P-M-G-L. One interpretation is that this genomicorganization reflects a common need among the mononegaviruses for largeamounts of the N and P proteins, a small amount of L, and intermediateamounts of M and G. Alternatively, it has been suggested that the lackof homologous recombination in mononegaviruses has resulted in theretention of an ancestral gene order that is not necessarily optimal forthe virus (Ball et al., J. Virol., 73:4705-4712, 1999; incorporatedherein by reference).

[0100] The constrained gene order and polar nature of transcription hasbeen proposed as an important factor in the regulation of geneexpression among mononegaviruses. However, other secondary factors arealso believed to affect the relative levels of expression of one or moreof the mononegaviral proteins, including differences in the efficienciesof cis-acting RNA signals, differences in efficiencies of translation ofvarious mRNAs, and differences in processing and stability of proteins.

[0101] The simple, prototypic mononegavirus, VSV, has 5 genes (Wagnerand Rose, Fields Virology, 1121-1136, 1996; Schubert et al., J. Virol.51:505-514, 1984; each incorporated herein by reference). However, othermononegaviruses have as many as 11 (RSV) or 12 (pneumonia virus of mice)genes (Barr et al., J. Virol., 68:5330-5334, 1994; incorporated hereinby reference). These include proteins such as the fusion F gene found inall paramyxoviruses and pneumoviruses, the C, D and V genes found insome paramyxoviruses, and the NS1, NS2, SH and M2 genes found in mostpneumoviruses. Also, even among the five proteins that may be common toVSV and RSV (N, P, M, G and L), there is clear sequence relatedness onlyfor L, and that relatedness is low (Poch et al., Embo. J. 8:3867-3674,1989; Stec et al., Virology 183:273-287, 1991; each incorporated hereinby reference).

[0102] Given this extensive difference in the array and structure ofgene products, taken together with differences in the structure andfunction of trans- and cis-acting components, the features andproperties of one mononegavirus, e.g., VSV, cannot be directlyextrapolated to other mononegaviruses, such as RSV. This uncertainty isexemplified by the finding that the RSV polymerase consists of 4 ratherthan 3 proteins, with the additional one being the M2-1 transcriptionantitermination factor that has no counterpart in VSV (Hardy and Wertz,J. Virol. 72:520-526, 1998; Hardy et al., J. Virol. 73:170-176, 1999;Collins et al., Proc. Natl. Acad. Sci. USA 93:81-85, 1996; Fearns andCollins, J. Virol. 73:5852-5864, 1999; each incorporated herein byreference). This proved to be critical for the efficient recovery ofrecombinant RSV (Collins et al., Virology 259:251-255, 1999;incorporated herein by reference). In addition, RSV RNA synthesis isfurther regulated by at least two proteins, NS1 and M2-2, which do nothave counterparts in VSV (Bermingham and Collins, Proc. Natl. Acad. Sci.USA 96:11259-64, 1999; Whitehead et al., J. Virol. 73:3438-3442, 1999;Atreya et al., J. Virol. 72:1452-61, 1998; Jin et al., J. Virol.74:74-82, 2000; each incorporated herein by reference).

[0103] A number of important features of VSV gene expression andregulation also do not appear to have any relevance to many othermononegaviruses and in particular to RSV, such as the involvement ofterminal complementarily in control of VSV gene expression (Wertz etal., Proc. Natl. Acad. Sci. USA 91:8587-8591, 1994; Whelan and Wertz, J.Virol. 73:297-306, 1999; Peeples and Collins, J. Virol. 74:146-155,2000; each incorporated herein by reference), the highly conserved VSVintergenic regions that are directly involved in gene expression (Barret al., J. Virol. 71:1794-1801, 1997; incorporated herein by reference)whereas those of RSV are not (Kuo et al., J. Virol. 70:6143-6150, 1996;incorporated herein by reference), a VSV genomic packaging signal(Whelan and Wertz, J. Virol. 73:307-315, 1999; incorporated herein byreference) not found in other mononegavirus groups, and a control of VSVgene expression and replication by N protein (Wagner and Rose, FieldsVirology, 1121-1136, 1996; Fearns et al., Virology 236:188-201, 1997;incorporated herein by reference). These features do not appear to occurin RSV (Peeples and Collins, J. Virol. 74:146-155, 2000; incorporatedherein by reference), while in contrast RSV has important features ofgene expression and regulation that do not occur in VSV, includingdivergent intergenic sequences that lack cis-acting functional elements(Kuo et al., J. Virol. 70:6143-6150, 1996; incorporated herein byreference), a gene overlap that mediates site-specific attenuation(Fearns and Collins, J. Virol. 73:388-397, 1999; Collins et al., Proc.Natl. Acad. Sci. USA 84:5134-5138, 1987; incorporated herein byreference), and the existence of a polymerase back-tracking mechanismcritical for expression of the L gene (Fearns and Collins, J. Virol.73:388-397, 1999; incorporated herein by reference). In particular, theexistence of a transcription antitermination factor that modulatessequential transcription (Fearns and Collins, J. Virol. 73:5852-5864,1999; incorporated herein by reference), specific regulatory proteins,site-specific attenuation, and gene-junction-specific modulation oftranscription (Hardy et al., J. Virol. 73:170-176, 1999; incorporatedherein by reference) contrast sharply with the situation with VSV.

[0104] In certain embodiments of the invention, gene position shifts areachieved by deletion of one or more of the RSV NS1, NS2, SH and/or Ggenes, which deletions are disclosed individually in theabove-incorporated references. Alternative gene or genome segmentdeletions can be constructed involving any of the above identified RSVgenes or genome segments, to alter gene position for remaining RSVgenes. In more detailed embodiments, multiple genes or genome segmentsare deleted, as exemplified in the above-incorporated references bypairwise deletion of the NS1 and NS2 genes (Bukreyev et al., J. Virol.71:8973-8982, 1997; Teng and Collins, J. Virol. 73:466-473, 1999;Whitehead et al., J. Virol. 73:3438-3442, 1999; incorporated herein byreference).

[0105] Deletion of one or more genes or genome segments within arecombinant RSV genome or antigenome has the effect of moving alldownstream genes closer to the promoter (e.g., by shifting thedownstream genes one or more gene positions in a promoter-proximaldirection). For example, the RSV NS1 and NS2 genes are the first andsecond genes in the genome map, and their coordinate deletion alters theposition of all of the remaining genes. Thus, when NS1 and NS2 aredeleted together, N is moved from position 3 to position 1, P fromposition 4 to position 2, and so on. Alternatively, deletion of anyother gene within the gene order will affect the position (relative tothe promoter) only of those genes which are located even furtherdownstream. For example, SH occupies position 6 in wild type virus, andits deletion does not affect M at position 5 (or any other upstreamgene) but moves G from position 7 to 6 relative to the promoter. Itshould be noted that gene deletion also can occur (rarely) inbiologically-derived virus. For example, a subgroup B RSV that had beenpassaged extensively in cell culture spontaneously deleted the SH and Ggenes (Karron et al., Proc. Natl. Acad. Sci. USA 94:13961-13966, 1997;incorporated herein by reference). Note that “upstream” and “downstream”refer to the promoter-proximal and promoter-distal directions,respectively (the promoter is at the 3′ leader end of negative-sensegenomic RNA).

[0106] A second example of gene order rearrangement useful within theinvention involves the insertion of a gene, genome segment orheterologous polynucleotide sequence into the recombinant RSV genome orantigenome to alter gene order or introduce a promoter-relative geneposition shift in the recombinant genome or antigenome (see, e.g.,Bukreyev et al., J. Virol. 70:6634-6641, 1996; Bukreyev et al., Proc.Natl. Acad. Sci. USA 96:2367-2372, 1999; Moriya et al., FEBS Lett.425:105-111, 1998; Singh and Billeter, J. Gen. Virol. 80:101-106, 1999;incorporated herein by reference). Each inserted gene displaces alldownstream genes by one position relative to the promoter. These andother displacement polynucleotides may be inserted or rearranged into anon-coding region (NCR) of the recombinant genome or antigenome, or maybe incorporated in the recombinant RSV genome or antigenome as aseparate gene unit (GU).

[0107] As used herein, “RSV gene” generally refers to a portion of theRSV genome encoding an mRNA and typically begins at the upstream endwith the 10-nucleotide gene-start (GS) signal and ends at the downstreamend with the 12 to 13-nucleotide gene-end (GE) signal. Ten such genesfor use within the invention are known for RSV, namely NS1, NS2, N, P,M, SH, G, F, M2 and L. The term “gene” is also used herein to refer to a“translational open reading frame” (ORF). ORF is more specificallydefined as a translational open reading frame encoding a significant RSVprotein, of which 11 are currently recognized: NS1, NS2, N, P, M, SH, G,F, M2-1 (alternatively, M2(ORF1)), M2-2 (alternatively, M2(ORF2)), andL. Thus, the term “gene” interchangeably refers to a genomic RNAsequence that encodes a subgenomic RNA, and to a ORF (the latter termapplies particularly in a situation such as in the case of the RSV M2gene, where a single MRNA contains two overlapping ORFs that encodedistinct proteins). Collins et al., J. Gen. Virol. 71:3015-3020, 1990;Bermingham and Collins, Proc. Natl. Acad. Sci. USA 96:11259-11264, 1999;Ahmadian et al., EMBO J. 19:2681-2689, 2000; Jin et al., J. Virol.74:74-82, 2000 (each incorporated herein by reference). When the term“gene” is used in the context of determining gene position relative to apromoter position, the term ordinarily refers strictly to anmRNA-encoding sequence bordered by transcription gene-start and gene-endsignal motifs (Collins et al., Proc. Natl. Acad. Sci. USA 83:4594-4598,1986; Kuo et al., J. Virol. 70:6892-6901, 1996; each incorporated hereinby reference).

[0108] By “genome segment” is meant any length of continuous nucleotidesfrom the RSV genome, which may be part of an ORF, a gene, or anextragenic region, or a combination thereof.

[0109] Genes and genome segments that may be selected for use asinserts, substitutions, deleted elements, or rearranged elements withingene position-shifted RSV of the invention include genes or genomesegments encoding a NS1, NS2, N, P, M, SH, M2(ORF1), M2(ORF2), L, F or Gprotein or portion thereof. Regulatory regions, such as the extragenicleader or trailer regions, can also be considered. In preferredembodiments of the invention, chimeric RSV incorporates one or moreheterologous gene(s) that encode an RSV F, G or SH glycoprotein.Alternatively, the recombinant RSV may incorporate a genome segmentencoding a cytoplasmic domain, transmembrane domain, ectodomain orimmunogenic epitope of a RSV F, G or SH glycoprotein. These immunogenicproteins, domains and epitopes are particularly useful within geneposition-shifted RSV because they generate novel immune responses in animmunized host. In particular, the G and F proteins, and immunogenicdomains and epitopes therein, provide major neutralization andprotective antigens. In addition, genes and genome segments encodingnon-RSV proteins, for example, an SH protein as found in mumps and SV5viruses, may be incorporated within gene position-shifted RSV of theinvention. Regulatory regions, such as the extragenic 3′ leader or 5′trailer regions, and gene-start, gene-end, intergenic regions, or 3′ or5′ non-coding regions, are also useful as heterologous (originating froma different RSV strain or subgroup or from a non-RSV source such as PIV,measles, mumps, etc.) substitutions or additions.

[0110] For example, addition or substitution of one or more immunogenicgene(s) or genome segment(s) from a human RSV subgroup or strain to orwithin a bovine recipient genome or antigenome yields a recombinant,chimeric virus or subviral particle capable of generating an immuneresponse directed against the human donor virus, including one or morespecific human RSV subgroups or strains, while the bovine backboneconfers an attenuated phenotype making the chimera a useful candidatefor vaccine development. In one such exemplary embodiment, one or morehuman RSV glycoprotein genes F, SH, and/or G are added to or substitutedwithin a partial or complete bovine genome or antigenome to yield anattenuated, infectious human-bovine chimera that elicits an anti-humanRSV immune response in a susceptible host. In other “chimeric”embodiments gene position-shifted RSV incorporate a heterologous gene orgenome segment encoding an immunogenic protein, protein domain orepitope from multiple human RSV strains, for example two F or G proteinsor immunogenic portions thereof from both RSV subgroups A and B. In yetadditional alternate embodiments a gene position-shifted RSV genome orantigenome encodes a chimeric glycoprotein in the recombinant virus orsubviral particle having both human and bovine glycoprotein domains orimmunogenic epitopes. For example, a heterologous genome segmentencoding a glycoprotein ectodomain from a human RSV F, SH or Gglycoprotein may be joined with a genome segment encoding correspondingbovine F, SH or G glycoprotein cytoplasmic and endodomains in thebackground bovine genome or antigenome.

[0111] According to the methods of the invention, human-bovine chimericRSV may be constructed by substituting the heterologous gene or genomesegment for a counterpart gene or genome segment in a partial RSVbackground genome or antigenome. Alternatively, the heterologous gene orgenome segment may be added as a supernumerary gene or genome segment incombination with a complete (or partial if another gene or genomesegment is deleted) RSV background genome or antigenome. For example,two human RSV G or F genes or genome segments can be included, one eachfrom RSV subgroups A and B.

[0112] Often, displacement genes or genome segments (includingheterologous genes or genome segments) are added at an intergenicposition within a partial or complete RSV genome or antigenome.Alternatively, the gene or genome segment can be placed in othernoncoding regions of the genome, for example, within the 5′ or 3′noncoding regions or in other positions where noncoding nucleotidesoccur within the partial or complete genome or antigenome. In oneaspect, noncoding regulatory regions contain cis-acting signals requiredfor efficient replication, transcription, and translation, and thereforerepresent target sites for modification of these functions byintroducing a displacement gene or genome segment or other mutation asdisclosed herein. In more detailed aspects of the invention, attenuatingmutations are introduced into cis-acting regulatory regions to yield,e.g., (1) a tissue specific attenuation (Gromeier et al., J. Virol.73:958-64, 1999; Zimmermann et al., J. Virol. 71:4145-9, 1997), (2)increased sensitivity to interferon (Zimmermann et al., J. Virol.71:4145-9, 1997), (3) temperature sensitivity (Whitehead et al.,Virology 247:232-9, 1998), (4) a general restriction in level ofreplication (Men et al., J. Virol. 70:3930-7, 1996; Muster et al., Proc.Natl. Acad. Sci. USA 88:5177-5181, 1991), and/or (5) host specificrestriction of replication (Cahour et al., Virology 207:68-76, 1995).These attenuating mutations can be achieved in various ways to producean attenuated gene position-shifted RSV of the invention, for example bypoint mutations, exchanges of sequences between related viruses, ordeletions.

[0113] In other alternative embodiments of the invention, geneposition-shifted RSV are provided wherein the recombinant RSV ismodified by deletion, insertion substitution, or rearrangement of aplurailty of genes or genome segments. In certain embodiments selected“gene sets” are coordinately transferred by one of these means into,within, or from the recombinant RSV genome or antigenome. Exemplary RSVgenes from which individual or coordinately transferred groups of genesmay be selected include the RSV N, P, NS1, NS2, M2-1 and M genes, whichmay be transferred singly or in any combination in a human or bovine RSVgenome or antigenome to yield an attenuated, gene-shifted derivative. Inmore detailed aspects, both N and P genes of a human or bovine RSV aredeleted, inserted, substituted or rearranged coordinately (e.g., bycoordinate deletion or substitution in a HRSV genome or antigenome bycounterpart N and P genes from a bovine RSV). This coordinate genetransfer is facilitated by functional cooperativity between certaingenes in the RSV genome, which often arises in the case of neighboringgene pairs in the genome. Thus, in other alternative embodiments, bothNS1 and NS2 genes are coordinately transferred, e.g., by substitution ina human RSV by counterpart NS1 and NS2 genes from a bovine RSV. In yetadditional embodiments, two or more of the M2- 1, M2-2 and L genes of aRSV are coordinately transferred. For certain vaccine candidates withinthe invention for which a high level of host-range restriction isdesired, each of the N, P, NS1, NS2, M2-1 and M genes of a human RSV arereplaced by counterpart N, P, NS 1, NS2, M2-1 and M genes from a bovineRSV.

[0114] Coordinate gene transfers within human-bovine chimeric RSV arealso directed to introduction of human antigenic genes within a bovinebackground genome or antigenome. In certain embodiments, one or morehuman RSV envelope-associated genes selected from F, G, SH, and M is/areadded or substituted within a partial or complete bovine RSV backgroundgenome or antigenome. For example, one or more human RSVenvelope-associated genes selected from F, G, SH, and M may be added orsubstituted within a partial bovine RSV background genome or antigenomein which one or more envelope-associated genes selected from F, G, SH,and M is/are deleted. In more detailed aspects, one or more genes from agene set defined as human RSV envelope-associated genes F, G, and M areadded within a partial bovine RSV background genome or antigenome inwhich envelope-associated genes F, G, SH, and M are deleted. Anexemplary human-bovine chimeric RSV bearing these features describe inthe examples below is rBRSV/A2-MGF.

[0115] In other aspects of the invention, insertion of heterologousnucleotide sequences into RSV vaccine candidates are employed separatelyto modulate the level of attenuation of candidate vaccine recombinants,e.g., for the upper respiratory tract. Thus, it is possible to insertnucleotide sequences into a rRSV that both direct the expression of aforeign protein and that attenuate the virus in an animal host, or touse nucleotide insertions separately to attenuate candidate vaccineviruses. General tools and methods for achieving these aspects of theinvention are provided, e.g., in U.S. Provisional Patent ApplicationSerial No. 60/170,195, U.S. patent application Ser. No. 09/458,813, andU.S. patent application Ser. No. 09/459,062 (each incorporated herein byreference). In one exemplary embodiment of the invention thus provided,insertion of the measles HA ORF between a selected RSV gene junctionwill restrict viral replication in vivo. In these aspects of theinvention, the selected gene insert may be relatively large(approximately 1900 nts or greater). In this context, size of the insertspecifies a selectable level of attenuation of the resulting recombinantvirus. Displacement sequences of various lengths derived from aheterologous virus, e.g., introduced as single gene units (GUs) anddesigned specifically to lack any significant ORF, reveal selectableattenuation effects due to increased genome length (i.e., versusexpression of an additional mRNA). Other constructs in which inserts ofsimilar sizes are introduced into a downstream noncoding region (NCR) ofa RSV gene are also useful within the invention.

[0116] To define some of the rules that govern the effect of geneinsertion on attenuation, gene units of varying lengths can be insertedinto a wild type RSV backbone and the effects of gene unit length onattenuation examined. Gene unit insertions engineered to not contain asignificant ORF permit evaluation of the effect of gene unit lengthindependently of an effect of the expressed protein of that gene. Theseheterologous sequences were inserted in a PIV backbone as an extra geneunit of sizes between 168 nt and 3918 nt between the HN and L genes. Inaddition, control cDNA constructions and viruses were made in whichinsertions of similar sizes were placed in the 3′-noncoding region ofthe HN gene of PIV and hence did not involve the addition of an extragene. These viruses were made to assess the effect of an increase in theoverall genome length and in gene number on attenuation. The insertionof an extra gene unit is expected to decrease the transcription of genesdownstream of the insertion site which will affect both the overallabundance and ratios of the expressed proteins. As demonstrated herein,gene insertions or extensions larger than about 3000 nts in lengthattenuated the wild type virus for the upper and lower respiratory tractof hamsters. Gene insertions of about 2000 nts in length furtherattenuated the rHPIV3cp45L vaccine candidate for the upper respiratorytract. Comparable gene insertions in RSV thus can have the dual effectof both attenuating a candidate vaccine virus and inducing a protectiveeffect against a second virus. Gene extensions in the 3′-noncodingregion of a gene, which cannot express additional proteins, can also beattenuating in and of themselves. Within these methods of the invention,gene insertion length is a determinant of attenuation.

[0117] A separate example of gene order rearrangement for use within theinvention involves changing the position of one or morenaturally-occurring genes relative to other naturally-occurring genes,without the introduction or deletion of substantial lengths ofpolynucleotides (e.g., greater than 100 nucleotides). For example, the Fand G genes of a human or human-bovine chimeric RSV can be shifted fromtheir natural gene order position to a more promotor proximal positionby excision and reinsertion of the genes into the recombinant genome orantigenome, without substantially altering the length of the recombinantRSV genome or antigenome.

[0118] These modifications in gene position and/or gene order typicallyresult in viruses with altered biological properties. For example,recombinant RSV of the invention lacking one or more selected genes, forexample NS1, NS2, SH, or G, NS1 and NS2 together, and SH and G together,may be attenuated in vitro, in vivo, or both. Whereas this phenotype islikely attributable primarily to the loss of expression of specificviral protein, it is also likely that the altered gene map contributedto the phenotype. This is supported by the results observed with theSH-deletion virus, which grew more efficiently than wild type in somecell types—probably due to an increase in the efficiency oftranscription, replication or both resulting from the gene deletion andresulting change in gene order and possibly genome size.

[0119] The ability to generate infectious RSV from cDNA provides amethod for introducing predetermined changes into infectious virus viathe cDNA intermediate. This method has been used to produce a series ofinfectious attenuated derivatives of wild type recombinant RSV strain A2that contain attenuating mutations including, for example, one or morenucleotide substitutions in cis-acting RNA signals and/or one or moreamino acid substitutions in one or more viral proteins and /or deletionof one or more genes or ablation its/their expression (Bukreyev et al.,J. Virol. 71:8973-8982, 1997; Whitehead et al., J. Virol. 72:4467-4471,1998; Whitehead et al., Virology 247:232-239, 1998; Bermingham andCollins, Proc. Natl. Acad. Sci. USA 96:11259-11264,1999; Juhasz et al.,Vaccine 17:1416-1424, 1999; Juhasz et al., J. Virol. 73:5176-5180, 1999;Teng and Collins, J. Virol. 73:466-473, 1999; Whitehead et al., J.Virol. 73:871-877, 1999; Whitehead et al., J. Virol. 73:3438-3442, 1999;Collins et al., Adv. Virus Res. 54:423-451, 1999; U.S. ProvisionalPatent Application No. 60/143,097, filed Jul. 9, 1999, U.S. patentapplication Ser. No. 09/611,829 and its corresponding PCT applicationpublished as WO 01/04321; each incorporated herein by reference).

[0120] Strain A2 represents antigenic subgroup A, and an effective RSVvaccine also should represent the other antigenic subgroup, subgroup B.The G and F glycoproteins are the major antigenic determinants and themajor protective RSV antigens (Connors et al., J. Virol. 65:1634-1637,1991; Murphy et al., Virus Research 32:13-36, 1994; Collins et al.,Fields Virology 2:1313-1352, 1996; and Crowe et al., New GenerationVaccines, 711-725, 1997; each incorporated herein by reference).Therefore, the G and F genes of recombinant strain A2 were replaced withtheir counterparts from the B1 strain of antigenic subgroup B (Whiteheadet al., J. Virol. 73:9773-9780, 1999, incorporated herein by reference).This was done using wild type and attenuated strain A2 backbones.Recombinant virus was obtained, and the “chimerization” did notdetectably interfere with virus replication. This demonstrated anexpedited method for making vaccine virus: specifically, to useattenuated strain A2 backbones to express the antigenic determinants ofsubgroup B (pending application). Thus, once an appropriately-attenuatedsubgroup A gene position-shifted RSV vaccine virus is identified inclinical trials, its backbone can be modified to produce a comparablesubgroup B vaccine in an expedited manner, and the two viruses can becombined to make a bivalent vaccine.

[0121] It has also been demonstrated in the above-incorporatedreferences that RSV useful within the invention can express a foreigngene added as an extra, supernumerary gene placed at any of a variety ofgenomic locations, preferably in an intergenic region. This concept hasbeen used to make a recombinant strain A2 virus that also expressed theG glycoprotein of subgroup B as a supernumerary gene. Thus, a singlevirus expressed antigenic determinants of the two subgroups. Anotherexample incorporated herein involves the expression of interferon gammaas an added gene, which resulted in attenuation without a reduction inimmunogenicity, and also provided a method to reduce the relative levelof stimulation of T helper lymphocyte subset 2, which has been proposedto mediate immunopathogenic responses to RSV (see, e.g., U.S.Provisional Application No. 60/143,425, filed Jul. 13, 1999, U.S. patentapplication Ser. No. 09/614,285 and its corresponding PCT applicationpublished as WO 01/04271, each incorporated herein by reference).

[0122] In alternate embodiments of the invention, a different basis forattenuation of a live virus vaccine incorporating a gene positionalshift is provided, which attenuation is based in part on host rangeeffects. In this regard, the instant disclosure provides attenuated,chimeric RSV by the introduction of genome segments, entire genes ormultiple genes between HRSV and BRSV. Host range differences betweenHRSV and BRSV are exemplified by the highly permissive growth of HRSV inchimpanzees compared to the barely detectable or undetectable growth ofBRSV in the same animal. The chimpanzee is a widely accepted model ofRSV infection and immunogenic activity in humans, exhibiting virusreplication and disease comparable to that of humans. As illustratedherein below, host range differences of chimeric RSV observed inchimpanzees are correlated with host range differences observed in cellculture, providing a convenient preliminary assay.

[0123] Host range effects observed in chimeric, human-bovine RSV of theinvention are generally related to nucleotide and amino acid sequencedifferences observed between HRSV and BRSV. For example, the percentamino acid identity between HRSV and BRSV for each of the followingproteins is: NS1 (69%), NS2 (84%), N (93%), P (81%), M (89%), SH (38%),G (30%), F (81%), M2-1 (80%), L (77%). Because of the extensive geneticdivergence between HRSV and BRSV (replacement of the N gene of HRSV withthat of BRSV, for example, involves approximately 26 amino aciddifferences), chimeric bovine-human RSV of the invention areparticularly useful vaccine candidates. As exemplified herein below,replacement of the BRSV G and F glycoproteins with those of HRSVincreases the permissivity of recombinant BRSV for replication inchimpanzees. The involvement of multiple genes and genome segments eachconferring multiple amino acid or nucleotide differences provides abroad basis for attenuation which is highly stable to reversion. Thismode of attenuation contrasts sharply to HRSV viruses attenuated by oneor several point mutations, where reversion of an individual mutationwill yield a significant or complete reacquisition of virulence. Inaddition, known attenuating point mutations in HRSV typically yield atemperature sensitive phenotype. This is because the temperaturesensitive phenotype was specifically used as the first screen toidentify altered progeny following exposure of HRSV to mutagens. Oneproblem with attenuation associated with temperature sensitivity is thatthe virus can be overly restricted for replication in the lowerrespiratory tract while being under attenuated in the upper respiratorytract. This is because there is a temperature gradient within therespiratory tract, with temperature being higher (and more restrictive)in the lower respiratory tract and lower (less restrictive) in the upperrespiratory tract. The ability of an attenuated virus to replicate inthe upper respiratory tract can result in complications includingcongestion, rhinitis, fever and otitis media. Thus, attenuation achievedsolely by temperature sensitive mutations may not be ideal. In contrast,host range mutations present in gene position-shifted RSV of theinvention will not in most cases confer temperature sensitivity.Therefore, this novel method of attenuation will (i) be more stablegenetically and phenotypically, and (ii) be less likely to be associatedwith residual virulence in the upper respiratory tract than other livevaccine approaches.

[0124] The amount of sequence divergence between BRSV and HRSV is abouttwice as much as between the HRSV A and B subgroups noted above. Thus,the F proteins have approximately 20% amino acid divergence between BRSVand HRSV, and the G proteins approximately 70% divergence (Lerch, etal., J. Virol. 64:5559-69, 1990; Lerch, et al., Virology 181:118-31,1991; Mallipeddi and Samal, J. Gen. Virol. 74:2001-4, 1993; Mallipeddiand Samal, Vet. Microbiol. 36:359-67, 1993; Samal et al., Virology180:453-456, 1991; Samal and Zamora, J. Gen. Virol. 72:1717-1720, 1991;Zamora and Samal, Virus Res. 24:115-121, 1992; ibid, J. Gen. Virol.73:737-741, 1992; Mallipeddi and Samal, J. Gen. Virol. 73:2441-2444,1992, Pastey and Samal, J. Gen. Virol. 76:193-197, 1995; Walravens etal., J. Gen. Virol. 71:3009-3014, 1990; Yunnus et al., J. Gen. Virol.79:2231-2238, 1998, each incorporated herein by reference).

[0125] In the prior disclosures incorporated herein, recombinant BRSVwas modified to replace the G and F BRSV genes with their human RSVcounterparts. The resulting chimeric BRSV/HRSV virus, bearing theantigenic determinants of human RSV on the BRSV backbone, replicatedmore efficiently in chimpanzees than did its BRSV parent, but remainedhighly attenuated. This indicated that the G and F genes contributed tothe host range restriction of BRSV, but showed that one or more othergenes also specified the host range restriction. This represents astarting point for constructing an optimal BRSV/HRSV chimeric virus thatfeatures a gene positional change as described above and which containsthe human RSV G and F antigenic determinants, wherein the resultingrecombinant RSV is attenuated by the presence of one or more BRSV genesto confer a host range restriction (Buchholz et al., J. Virol.74:1187-1199, 2000; U.S. patent application Ser. No. 60/143,132, filedJul. 9, 1999; each incorporated herein by reference).

[0126] Detailed descriptions of the materials and methods for producingrecombinant RSV from cDNA, and for making and testing the full range ofmutations and nucleotide modifications disclosed herein as supplementalaspects of the present invention, are set forth in, e.g., U.S.Provisional Patent Application No. 60/007,083, filed Sep. 27, 1995; U.S.patent application Ser. No. 08/720,132, filed Sep. 27, 1996; U.S.Provisional Patent Application No. 60/021,773, filed Jul. 15, 1996 andU.S. patent application Ser. No. 08/892,403, now issued as U.S. Pat. No.5,993,824; U.S. Provisional Patent Application No. 60/046,141, filed May9, 1997; U.S. Provisional Patent Application No. 60/047,634, filed May23, 1997; U.S. Pat. No. 5,993,824, issued Nov. 30, 1999 (correspondingto International Publication No. WO 98/02530); U.S. patent applicationSer. No. 09/291,894, filed by Collins et al. on Apr. 13, 1999 andcorresponding to published PCT application WO 00/61737; U.S. ProvisionalPatent Application No. 60/129,006, filed by Murphy et al. on Apr. 13,1999; Crowe et al., Vaccine 12: 691-699, 1994; and Crowe et al., Vaccine12: 783-790, 1994; Collins, et al., Proc Nat. Acad. Sci. USA92:11563-11567, 1995; Bukreyev, et al., J Virol 70:6634-41, 1996, Juhaszet al., J. Virol. 71(8):5814-5819, 1997; Durbin et al., Virology235:323-332, 1997; Karron et al., J. Infect. Dis. 176:1428-1436, 1997;He et al. Virology 237:249-260, 1997; Baron et al. J. Virol.71:1265-1271, 1997; Whitehead et al., Virology 247(2):232-9, 1998a;Whitehead et al., J. Virol. 72(5):4467-4471, 1998b; Jin et al. Virology251:206-214, 1998; Bukreyev, et al., Proc. Nat. Acad. Sci. USA96:2367-2372, 1999; Bermingham and Collins, Proc. Natl. Acad. Sci. USA96:11259-11264, 1999 Juhasz et al., Vaccine 17:1416-1424, 1999; Juhaszet al., J. Virol. 73:5176-5180, 1999; Teng and Collins, J. Virol.73:466-473, 1999; Whitehead et al., J. Virol. 73:9773-9780, 1999;Whitehead et al., J. Virol. 73:871-877, 1999; and Whitehead et al., J.Virol. 73:3438-3442, 1999.

[0127] Exemplary methods for producing recombinant RSV from cDNA involveintracellular coexpression, typically from plasmids cotransfected intotissue culture cells, of an RSV antigenomic RNA and the RSV N, P, M2-1and L proteins. This launches a productive infection that results in theproduction of infectious cDNA-derived virus, which is termed recombinantvirus. Once generated, recombinant RSV is readily propagated in the samemanner as biologically-derived virus, and a recombinant virus and acounterpart biologically-derived virus cannot be distinguished unlessthe former had been modified to contain one or more introduced changesas markers.

[0128] In more detailed aspects, the foregoing incorporated documentsdescribe methods and procedures useful within the invention formutagenizing, isolating and characterizing RSV to obtain attenuatedmutant strains (e.g., temperature sensitive (ts), cold passaged (cp)cold-adapted (ca), small plaque (sp) and host-range restricted (hr)mutant strains) and for identifying the genetic changes that specify theattenuated phenotype. In conjunction with these methods, the foregoingdocuments detail procedures for determining replication, immunogenicity,genetic stability and protective efficacy of biologically derived andrecombinantly produced attenuated human RSV, including human RSV A and Bsubgroups, in accepted model systems, including murine and non-humanprimate model systems. In addition, these documents describe generalmethods for developing and testing immunogenic compositions, includingmonovalent and bivalent vaccines, for prophylaxis and treatment of RSVinfection.

[0129] The ability to generate infectious RSV from cDNA provides amethod for introducing predetermined changes into infectious virus viathe cDNA intermediate. This method has been demonstrated to produce awide range of infectious, attenuated derivatives of RSV, for examplerecombinant vaccine candidates containing one or more amino acidsubstitutions in a viral protein, deletion of one or more genes orablation of gene expression, and/or one or more nucleotide substitutionsin cis-acting RNA signals yielding desired effects on viral phenotype(see, e.g., Bukreyev et al., J. Virol. 71:8973-8982, 1997; Whitehead etal., J. Virol. 72:4467-4471, 1998; Whitehead et al., Virology247:232-239, 1998; Bermingham and Collins, Proc. Natl. Acad. Sci. USA96:11259-11264,1999; Juhasz et al., Vaccine 17:1416-1424, 1999; Juhaszet al., J. Virol. 73:5176-5180, 1999; Teng and Collins, J. Virol.73:466-473, 1999; Whitehead et al., J. Virol. 73:871-877, 1999;Whitehead et al., J. Virol. 73:3438-3442, 1999; and Collins et al., Adv.Virus Res. 54:423-451, 1999, each incorporated herein by reference).

[0130] Exemplary of the foregoing teachings are methods for constructingand evaluating infectious recombinant RSV modified to incorporatephenotype-specific mutations identified in biologically-derived RSVmutants, e.g., cp and ts mutations adopted in recombinant RSV frombiologically derived designated cpts RSV 248 (ATCC VR 2450), cpts RSV248/404 (ATCC VR 2454), cpts RSV 248/955 (ATCC VR 2453), cpts RSV 530(ATCC VR 2452), cpts RSV 530/1009 (ATCC VR 2451), cpts RSV 530/1030(ATCC VR 2455), RSV B-1 cp52/2B5 (ATCC VR 2542), and RSV B-1 cp-23 (ATCCVR 2579). These methods are readily adapted for construction ofrecombinant gene position-shifted RSV of the invention. The recombinantRSV thus provided may incorporate two or more ts mutations from thesame, or different, biologically derived RSV mutant(s), for example oneor more of the 248/404, 248/955, 530/1009, or 530/1030 biologicalmutants. In the latter context, multiply attenuated recombinants mayhave a combination of attenuating mutations from two, three or morebiological mutants, e.g., a combination of attenuating mutations fromthe RSV mutants 530/1009/404, 248/404/1009, 248/404/1030, or248/404/1009/1030 mutants. In exemplary embodiments, one or moreattenuating mutations specify a temperature-sensitive substitution atamino acid Asn43, Phe521, Gln831, Met1169, or Tyr1321 in the RSVpolymerase gene or a temperature-sensitive nucleotide substitution inthe gene-start sequence of gene M2. Preferably, these mutations involveidentical or conservative changes with the following changes identifiedin biologically derived mutant RSV, for example changes conservative tothe following substitutions identified in the L polymerase gene: Ile forAsn43, Leu for Phe521, Leu for Gln831, Val for Met1169, and Asn forTyr1321.

[0131] Yet additional mutations that may be incorporated in geneposition-shifted RSV of the invention are mutations, e.g., attenuatingmutations, identified in heterologous RSV or more distantly relatednegative stranded RNA viruses. In particular, attenuating and otherdesired mutations identified in one negative stranded RNA virus may be“transferred”, e.g., copied, to a corresponding position within a humanor bovine RSV genome or antigenome, either within the geneposition-shifted RSV or as a means of constructing the geneposition-shifted RSV. Briefly, desired mutations in one heterologousnegative stranded RNA virus are transferred to the RSV recipient (e.g.,bovine or human RSV, respectively). This involves mapping the mutationin the heterologous virus, thus identifying by sequence alignment thecorresponding site in human or bovine RSV, and mutating the nativesequence in the RSV recipient to the mutant genotype (either by anidentical or conservative mutation), as described in U.S. ProvisionalPatent Application No. 60/129,006, filed by Murphy et al. on Apr. 13,1999, incorporated herein by reference. As this disclosure teaches, itis preferable to modify the chimeric genome or antigenome to encode analteration at the subject site of mutation that correspondsconservatively to the alteration identified in the heterologous mutantvirus. For example, if an amino acid substitution marks a site ofmutation in the mutant virus compared to the corresponding wild-typesequence, then a similar substitution should be engineered at thecorresponding residue(s) in the recombinant virus. Preferably thesubstitution will involve an identical or conservative amino acid to thesubstitute residue present in the mutant viral protein. However, it isalso possible to alter the native amino acid residue at the site ofmutation non-conservatively with respect to the substitute residue inthe mutant protein (e.g., by using any other amino acid to disrupt orimpair the function of the wild-type residue). Negative stranded RNAviruses from which exemplary mutations are identified and transferredinto a gene position-shifted RSV of the invention include other RSVs(e.g., murine), PIV, Sendai virus (SeV), Newcastle disease virus (NDV),simian virus 5 (SV5), measles virus (MeV), rindepest virus, caninedistemper virus (CDV), rabies virus (RaV) and vesicular stomatitis virus(VSV). A variety of exemplary mutations are disclosed, including but notlimited to an amino acid substitution of phenylalanine at position 521of the RSV L protein (corresponding to a substitution of phenylalanineat position 456 of the HPIV3 L protein). In the case of mutations markedby deletions or insertions, these can be introduced as correspondingdeletions or insertions into the recombinant virus, however theparticular size and amino acid sequence of the deleted or insertedprotein fragment can vary.

[0132] A variety of additional types of mutations are also disclosed inthe foregoing incorporated references and can be readily engineered intoa recombinant gene position-shifted RSV of the invention to calibrateattenuation, immunogenicity or provide other advantageous structuraland/or phenotypic effects. For example, restriction site markers areroutinely introduced within the gene position-shifted genome orantigenome to facilitate cDNA construction and manipulation. Alsodescribed in the incorporated references are a wide range of nucleotidemodifications other than point or site-specific mutations that areuseful within the instant invention. For example, methods andcompositions are disclosed for producing recombinant RSV expressing anadditional foreign gene, e.g., a chloramphenicol acetyl transferase(CAT) or luciferase gene. Such recombinants generally exhibit reducedgrowth associated with the inserted gene. This attenuation appears toincrease with increasing length of the inserted gene. The finding thatinsertion of a foreign gene into recombinant RSV reduces level ofreplication and is stable during passage in vitro provides anothereffective method for attenuating RSV for vaccine use. Similar orimproved effects can thus be achieved by insertion of other desiredgenes, for example cytokines such as interferon-y, interleukin-2,interleukin-4 and GM-CSF, among others.

[0133] Within the methods of the invention, additional genes or genomesegments may be inserted into or proximate to the gene position-shiftedRSV genome or antigenome. These genes may be under common control withrecipient genes, or may be under the control of an independent set oftranscription signals. Genes of interest include the RSV genesidentified above, as well as non-RSV genes. Non-RSV genes of interestinclude those encoding cytokines (e.g., IL-2 through IL-18, especiallyIL-2, IL-6 and IL-12, IL-18, etc.), gamma-interferon, and proteins richin T helper cell epitopes. These additional proteins can be expressedeither as a separate protein, or as a chimera engineered from a secondcopy of one of the RSV proteins, such as SH. This provides the abilityto modify and improve the immune responses against RSV bothquantitatively and qualitatively.

[0134] Increased genome length results in attenuation of the resultantRSV, dependent in part upon the length of the insert. In addition, theexpression of certain proteins, e.g. a cytokine, from a non-RSV geneinserted into gene position-shifted RSV will result in attenuation ofthe virus due to the action of the protein. Exemplary cytokines thatyield an infectious, attenuated viral phenotype and high level cytokineexpression from RSV transfected cells include interleukin-2 (IL-2),IL-4, GM-CSF, and γ-interferon. Additional effects includingaugmentation of cellular and or humoral immune responses will alsoattend introduction of cytokines into gene position-shifted RSV of theinvention.

[0135] Deletions, insertions, substitutions and other mutationsinvolving changes of whole viral genes or genome segments within geneposition-shifted RSV of the invention yield highly stable vaccinecandidates, which are particularly important in the case ofimmunosuppressed individuals. Many of these changes will result inattenuation of resultant vaccine strains, whereas others will specifydifferent types of desired phenotypic changes. For example, accessory(i.e., not essential for in vitro growth) genes are excellent candidatesto encode proteins that specifically interfere with host immunity (see,e.g., Kato et al., EMBO. J. 16:578-87, 1997, incorporated herein byreference). Ablation of such genes in chimeric vaccine viruses isexpected to reduce virulence and pathogenesis and/or improveimmunogenicity.

[0136] Additional, independent nucleotide modifications disclosed in theforegoing references for incorporation into recombinant geneposition-shifted RSV of the invention include partial or completedeletion or ablation of a selected RSV gene. Thus, RSV genes or genomesegments may be deleted, including partial or complete deletions of openreading frames and/or cis-acting regulatory sequences of the RSV NS1,NS2, N, P, M, G, F, SH, M2(ORF1), M2(ORF2) and/or L genes. In oneexample, a recombinant RSV was generated in which expression of the SHgene was been ablated by removal of a polynucleotide sequence encodingthe SH mRNA and protein. Deletion of the SH gene yielded not onlyrecoverable, infectious RSV, but one which exhibited substantiallyimproved growth in tissue culture based on both yield of infectiousvirus and plaque size. This improved growth in tissue culture specifiedby the SH deletion provides useful tools for developing geneposition-shifted RSV vaccines, for example by overcoming problems ofpoor RSV yields in culture. Moreover, these deletions are highly stableagainst genetic reversion, rendering RSV clones derived therefromparticularly useful as vaccine agents.

[0137] SH-minus RSV recombinants also exhibit site-specific attenuationin the upper respiratory tract of mice, which presents novel advantagesfor vaccine development. Current RSV strains under evaluation as livevirus vaccines, for example cp mutants, do not exhibit significantlyaltered growth in tissue culture. These are host range mutations andthey restrict replication in the respiratory tract of chimpanzees andhumans approximately 100-fold in the lower respiratory tract. Anotherexemplary type of mutation, ts mutations, tend to preferentiallyrestrict virus replication in the lower respiratory tract due to thegradient of increasing body temperature from the upper to the lowerrespiratory tract. In contrast to these cp and ts mutants, SH-minus RSVmutants have distinct phenotypes of greater restriction in the upperrespiratory tract. This is particularly desirable for vaccine virusesfor use in very young infants, because restriction of replication in theupper respiratory tract is required to ensure safe vaccineadministration in this vulnerable age group whose members breathepredominantly through the nose. Further, in any age group, reducedreplication in the upper respiratory tract will reduce morbidity fromotitis media. In addition to these advantages, the nature of SH deletionmutations, involving e.g., nearly 400 nt and ablation of an entire MRNA,represents a type of mutation which will be highly refractory toreversion. The utility of the SH-minus deletion as a “displacementpolynucleotide” for simultaneously directing a gene position-shift,e.g., to upregulate F and G glycoprotein gene expression, is amplyevinced in the examples below.

[0138] Also discussed in the context of SH gene modifications is acomparison of SH genes among different RSVs, including human and bovineRSVs, and other pneumoviruses to provide additional tools and methodsfor generating useful RSV recombinant vaccines. For example, the two RSVantigenic subgroups, A and B, exhibit a relatively high degree ofconservation in certain SH domains. In two such domains, the N-terminalregion and putative membrane-spanning domains of RSV A and B display 84%identity at the amino acid level, while the C-terminal putativeectodomains are more divergent (approx. 50% identity). Comparison of theSH genes of two human RSV subgroup B strains, 8/60 and 18537, identifiedonly a single amino acid difference (Anderson et al., supra). The SHproteins of human versus bovine RSV are approximately 40% identical, andshare major structural features including (i) an asymmetric distributionof conserved residues; (ii) very similar hydrophobicity profiles; (iii)the presence of two N-linked glycosylation sites with one site being oneach side of the hydrophobic region; and (iv) a single cysteine residueon the carboxyterminal side of the central hydrophobic region of each SHprotein. (Anderson et al., supra). By evaluating these and othersequence similarities and differences, selections can be made ofheterologous sequence(s) that can be substituted or inserted withininfectious gene position-shifted RSV clones, for example to yieldvaccines having multi-specific immunogenic effects or, alternatively orin addition, desirable effects such as attenuation.

[0139] In alternate embodiments of the invention, partial gene deletionsor other limited nucleotide deletions are engineered into recombinantRSV to yield desired phenotypic changes. In one example, the length ofthe RSV genome is reduced by deleting sequence from the downstreamnoncoding region of the SH gene. This exemplary partial gene deletionwas constructed using a version of the antigenome cDNA containing anXmaI site in the G-F intergenic region, a change which of itself wouldnot be expected to affect the encoded virus. The encoded virus,designated RSV/6120, has silent nucleotide substitutions in the lastthree codons and termination codon of the SH ORF and has a deletion of112 nucleotides from the SH downstream non-translated region (positions4499-4610 in the recombinant antigenome). This deletion leaves thegene-end signal (Bukreyev, et al., J. Virol., 70:6634-41, 1996,incorporated herein by reference) intact. These point mutations and112-nt deletion do not alter the encoded amino acids of any of the viralproteins, nor do they interrupt any of the known viral RNA signals orchange the number of encoded mRNAs.

[0140] The 6120 virus was analyzed for the efficiency of multi-stepgrowth in parallel with its full-length counterpart, D53, and showed apeak titer that was reproducibly higher than that of the D53 virus by afactor of 1.5- to 2-fold. Thus, the small, partial deletion in the SHgene of a 112-nt noncoding sequence resulted in a substantial increasein growth efficiency in vitro.

[0141] Other partial gene deletions and small nucleotide deletions canbe readily engineered in recombinant RSV of the invention to alter viralphenotype, including nucleotide deletions in: (1) nontranslated sequenceat the beginning and/or end of the various ORFs apart from thecis-acting RNA signal, (2) intergenic regions, and (3) the regions ofthe 3′leader and 5′ trailer that are not essential for promoteractivity. Examples of nontranslated gene sequence for deletion orinsertion include the following regions of the downstream untranslatedregion of the NS1, NS2, P, M, F, and M2 genes: namely, sequencepositions 519-563, 1003-1086, 3073-3230, 4033-4197, 7387-7539 and8433-8490, respectively, numbered according to the recombinantantigenome. Also, as additional examples, nt 55-96, nt 606-624, nt4231-4300 can be deleted from the upstream nontranslated region of theNS1, NS2 and SH genes respectively. Any partial or complete deletion inone or more of these sequences can be achieved in accordance with theteachings herein to provide candidates that are readily screened forbeneficial phenotypic changes specified by selected deletions.Additional nontranslated regions within the RSV genome are also usefulin this regard. Since the gene-start and gene-end signals have beenmapped and characterized with regard to important positions (Kuo, etal., J. Virol., 71:4944-4953; 1997; Harmon, et al., J. Virol., 75:36-44,2001, each incorporated herein by reference), deletions or modificationsthat involve one or a few (e.g., 3-10, 10-20, 20-45) nt can beconsidered. In some cases, specific additional advantages may beobtained. For example, in the G gene, deletion of nt 4683 to 4685, whichincludes one nt of the gene-start signal and two nt of nontranslatedsequence, ablates the first AUG in the mRNA, which does not initiate asignificant ORF but is thought to divert ribosomes from the next AUGwhich initiates the G ORF. In addition, this deletion restores the GSsignal and retains the translation start site of the G ORF. Thus,nontranslated sites for modification can be selected based on knowledgeof the genome, or can be selected at random and tested expeditiously bythe methods of the present invention.

[0142] With regard to intergenic sequences, studies with minigenomesshow that an intergenic region can be reduced to a single nt or deletedaltogether without affecting transcription and RNA replication. Theintergenic regions of strain A2 represent another 207 nt in aggregate(noting that the NS2-N intergenic region of the recombinant antigenomewas engineered to be 1 nt longer than its biological equivalent; see,e.g., Collins, et al., Proc. Natl. Acad. Sci. USA, 92:11563-11567, 1995,incorporated herein by reference).

[0143] The RSV 5′ trailer region is 155 nt in length and thus isapproximately 100 nt longer than the corresponding region of mostmononegaviruses and is 111 nt longer than the RSV leader region. Studieswith minigenomes suggest that much of this sequence is not essential andis a candidate for modification (Kuo, et al., J. Virol., 70:6892-901,1996, incorporated herein by reference). For example, the region oftrailer that immediately follows the L gene could be reduced in size by75 nt, 100 nt, 125 nt or more, leaving intact the 5′ genomic terminus(which encodes the 3′ end of the antigenome, including the antigenomepromoter). Similarly, the 44-nt leader region might be modified. Forexample, the first 11 nt at the 3′ leader end form the core of the viralpromoter, and thus sequence from the remainder of the leader regionmight be deleted or otherwise modified.

[0144] In certain embodiments of the invention, deleting one or more ofthe nontranslated sequences (partially or completely) described abovefor the NS1, NS2, SH, F and M2 genes will result in an adjustablereduction in genome length of up to 806 nt—more than 7-fold greater thanthe 112-nt deletion described in the instant example. Deleting partiallyor completely one or more of the intergenic regions between the firstnine genes (e.g., down to a minimal length of one nt each) would yieldup to an additional 198 nt of adjustable deletion. Partial or completedeletions from the trailer and/or leader can yield up to 50, 75, 100, ormore nt in additional deletion. Thus, for example, combining 806 nt fromnontranslated gene sequence with 198 nt from the intergenic regions and100 nt from the trailer yields 1104 nt in aggregate, representing nearlya 10-fold greater deletion than 112-nt deletion described here (andrepresenting more than 7% of the RSV genome).

[0145] In another example described in the above-incorporatedreferences, expression of the NS2 gene is ablated by introduction ofstop codons into the translational open reading frame (ORF). The rate ofrelease of infectious virus was reduced for this NS2 knock-out viruscompared to wild-type. In addition, comparison of the plaques of themutant and wild-type viruses showed that those of the NS2 knock-out weregreatly reduced in size. This type of mutation can thus be incorporatedwithin viable recombinant gene position-shifted RSV to yield alteredphenotypes, in this case reduced rate of virus growth and reduced plaquesize in vitro. These and other knock-out methods and mutants willtherefore provide for yet additional recombinant RSV vaccine agents,based on the known correlation between reduced plaque size in vitro andattenuation in vivo. Expression of the NS2 gene also was ablated bycomplete removal of the NS2 gene, yielding a virus with a similarphenotype.

[0146] Other RSV genes which have been successfully deleted include theNS1 and M2-2 genes. The former was deleted by removal of thepolynucleotide sequence encoding the respective protein, and the latterby introducing a frame-shift or altering translational start sites andintroducing stop codons. Interestingly, recovered NS1-minus virusproduce small plaques in tissue culture albeit not as small as those ofthe NS2 deletion virus. The fact that the NS1-minus virus can grow,albeit with reduced efficiency, identifies the NS1 protein as anaccessory protein, one that is dispensable to virus growth. The plaquesize of the NS1 -minus virus was similar to that of NS2 knock-out virusin which expression of the NS2 protein was ablated by introducingtranslational stop codons into its coding sequence The small plaquephenotype is commonly associated with attenuating mutations. This typeof mutation can thus be independently incorporated within viablerecombinant RSV to yield altered phenotypes. These and other knock-outmethods and mutants will therefore provide for yet additionalrecombinant gene position-shifted RSV vaccine agents, based on the knowncorrelation between plaque size in vitro and attenuation in vivo. TheNS2 knock-out mutant exhibited a moderately attenuated phenotype in theupper respiratory tract and a highly attenuated phenotype in the lowerrespiratory tract in naive chimpanzees. This mutant also elicitedgreatly reduced disease symptoms in chimps while stimulating significantresistance to challenge by the wild-type virus (Whitehead et al., J.Virol. 73:3438-3442, 1999, incorporated herein by reference).

[0147] Yet additional methods and compositions provided within theincorporated references and useful within the invention involvedifferent nucleotide modifications within gene position-shifted RSV thatalter cis-acting regulatory sequences within the chimeric genome orantigenome. For example, a translational start site for a secreted formof the RSV G glycoprotein can be deleted to disrupt expression of thisform of the G glycoprotein. The RSV G protein is synthesized in twoforms: as an anchored type II integral membrane protein and as aN-terminally resected form which lacks essentially all of the membraneanchor and is secreted (Hendricks et al., J. Virol. 62:2228-2233, 1988).The two forms have been shown to be derived by translational initiationat two different start sites: the longer form initiates at the first AUGof the G ORF, and the second initiates at the second AUG of the ORF atcodon 48 and is further processed by proteolysis (Roberts et al., J.Virol. 68: 4538-4546, 1994). The presence of this second start site ishighly conserved, being present in all strains of human, bovine andovine RSV sequenced to date. It has been suggested that the soluble formof the G protein might mitigate host immunity by acting as a decoy totrap neutralizing antibodies. Also, soluble G has been implicated inpreferential stimulation of a Th2-biased response, which in turn appearsto be associated with enhanced immunopathology upon subsequent exposureto RSV. With regard to an RSV vaccine virus, it is highly desirable tominimize antibody trapping or imbalanced stimulation of the immunesystem, and so it would be desirable to ablate expression of thesecreted form of the G protein. This has been achieved in recombinantvirus. Thus, this mutation is particularly useful to qualitativelyand/or quantitatively alter the host immune response elicited by therecombinant virus, rather than to directly attenuate the virus.

[0148] The incorporated references also describe modulation of thephenotype of recombinant RSV by altering cis-acting transcriptionsignals of exemplary genes, e.g., NS1 and NS2. The results of thesenucleotide modifications are consistent with modification of geneexpression by altering cis-regulatory elements, for example to decreaselevels of readthrough mRNAs and increase expression of proteins fromdownstream genes. The resulting recombinant viruses will preferablyexhibit increased growth kinetics and increased plaque size. Exemplarymodifications to cis-acting regulatory sequences include modificationsto gene end (GE) and gene start (GS) signals associated with RSV genes.In this context, exemplary changes include alterations of the GE signalsof the NS1 and NS2 genes rendering these signals identical to thenaturally-occurring GE signal of the RSV N gene. The resultingrecombinant virus exhibits increased growth kinetics and plaque size andtherefore provide yet additional means for beneficially modifyingphenotypes of gene position-shifted RSV vaccine candidates.

[0149] Methods and compositions provided in the above-incorporatedreferences also allow production of attenuated gene position-shifted RSVvaccine viruses comprising sequences from both RSV subgroups A and B,e.g., to yield a RSV A or B vaccine or a bivalent RSV A/B vaccine (see,e.g., U.S. Patent Application No. 09/291,894, filed by Collins et al. onApr. 13, 1999, incorporated herein by reference). Further augmenting theinvention in this context, specific attenuating mutations have beenincorporated into chimeric RSV A/B viruses include: (i) three of thefive cp mutations, namely the mutation in N (V267I) and the two in L(C319Y and H1690Y), but not the two in F since these are removed bysubstitution with the B1 F gene; (ii) the 248 (Q831L), 1030 (Y1321N)and, optionally, 404-L (Dl 183E) mutations which have been identified inattenuated strain A2 viruses; (iii) the single nucleotide substitutionat position 9 in the gene-start signal of the M2 gene, and (iv) deletionof the SH gene. Other immediately available mutations in geneposition-shifted RSV carrying RSV A and or RSV B genes or genomesegments include, but are not limited to, NS1, NS2, G, or M2-2 genedeletions, and the 530 and 1009 mutations, alone or in combination.

[0150] In other detailed aspects of the invention, gene position-shiftedRSV are employed as “vectors” for protective antigens of heterologouspathogens, including other RSVs and non-RSV viruses and non-viralpathogens. Within these aspects, the gene position-shifted RSV genome orantigenome comprises a partial or complete RSV “vector genome orantigenome” combined with one or more heterologous genes or genomesegments encoding one or more antigenic determinants of one or moreheterologous pathogens (see, e.g., U.S. Provisional Patent ApplicationSer. No. 60/170,195; U.S. patent application Ser. No. 09/458,813; andU.S. patent application Ser. No. 09/459,062, each incorporated herein byreference). The heterologous pathogen in this context may be aheterologous RSV (e.g., a different RSV strain or subgroup) and theheterologous gene(s) or genome segment(s) can be selected to encode oneor more of the above identified RSV proteins, as well as proteindomains, fragments, and immunogenic regions or epitopes thereof. RSVvector vaccines thus constructed may elicit a polyspecific immuneresponse and may be administered simultaneously or in a coordinateadministration protocol with other vaccine agents.

[0151] Gene position-shifted RSV engineered as vectors for otherpathogens may comprise a vector genome or antigenome that is a partialor complete HRSV genome or antigenome, which is combined with or ismodified to incorporate one or more heterologous genes or genomesegments encoding antigenic determinant(s) of one or more heterologousRSV(s), including heterologous HRSVs selected from HRSV A or HRSV B. Inalternative aspects, the vector genome or antigenome is a partial orcomplete HRSV genome or antigenome and the heterologous gene(s) orgenome segment(s) encoding the antigenic determinant(s) is/are of one ormore non-RSV pathogens. The vector genome or antigenome may furtherincorporate one or more gene(s) or genome segment(s) of a BRSV thatspecifies attenuation. Alternatively, the vector virus may be comprise apartial or complete BRSV background genome or antigenome incorporatingone or more HRSV genes or genome segments, wherein the geneposition-shifted RSV vector virus is modified to include one or moredonor gene(s) or genome segment(s) encoding an antigenic determinant ofa non-RSV pathogen.

[0152] Thus, in certain detailed aspects of the invention, geneposition-shifted RSV are provided as vectors for a range of non-RSVpathogens (see, e.g., U.S. Provisional Patent Application Serial No.60/170,195; U.S. patent application Ser. No. 09/458,813; and U.S. patentapplication Ser. No. 09/459,062, each incorporated herein by reference).The vector genome or antigenome for use within these aspects of theinvention may comprise a partial or complete BRSV or HRSV genome orantigenome incorporating, respectively, a heterologous HRSV or BRSV geneor genome segment, and the heterologous pathogen may be selected frommeasles virus, subgroup A and subgroup B respiratory syncytial viruses,HPIV1, HPIV2, HPIV3, mumps virus, human papilloma viruses, type 1 andtype 2 human immunodeficiency viruses, herpes simplex viruses,cytomegalovirus, rabies virus, Epstein Barr virus, filoviruses,bunyaviruses, flaviviruses, alphaviruses and influenza viruses.

[0153] For example, a HRSV or BRSV vector genome or antigenome forconstructing gene position-shifted RSV of the invention may incorporateheterologous antigenic determinant(s) selected from the measles virus HAand F proteins, or antigenic domains, fragments and epitopes thereof. Inexemplary embodiments, a transcription unit comprising an open readingframe (ORF) of a measles virus HA gene is added to or incorporatedwithin a BRSV or HRSV3 vector genome or antigenome. Alternatively geneposition-shifted RSV of the invention may used as vectors to incorporateheterologous antigenic determinant(s) from a parainfluenza virus (PIV),for example by incorporating one or more genes or genome segments thatencode(s) a HPIV1, HPIV2, or HPIV3 HN or F glycoprotein or immunogenicdomain(s) or epitope(s) thereof.

[0154] The introduction of heterologous immunogenic proteins, domainsand epitopes within gene position-shifted RSV is particularly useful togenerate novel immune responses in an immunized host. For example,addition or substitution of an immunogenic gene or genome segment fromone, donor RSV subgroup or strain within a recipient genome orantigenome of a different RSV subgroup or strain can generate an immuneresponse directed against the donor subgroup or strain, the recipientsubgroup or strain, or against both the donor and recipient subgroup orstrain. To achieve this purpose, gene position-shifted RSV may also beconstructed that express a chimeric protein, e.g., an immunogenicglycoprotein having a cytoplasmic tail and/or transmembrane domainspecific to one RSV fused to an ectodomain of a different RSV toprovide, e.g., a human-bovine fusion protein, or a fusion proteinincorporating domains from two different human RSV subgroups or strains.In a preferred embodiment, a gene position-shifted RSV genome orantigenome encodes a chimeric glycoprotein in the recombinant virus orsubviral particle having both human and bovine glycoprotein domains orimmunogenic epitopes. For example, a heterologous genome segmentencoding a glycoprotein ectodomain from a human RSV F, SH or Gglycoprotein may be joined with a polynucleotide sequence (i.e., agenome segment) encoding the corresponding bovine F, SH or Gglycoprotein cytoplasmic and endo domains to form the geneposition-shifted RSV genome or antigenome.

[0155] In other embodiments, gene position-shifted RSV useful in avaccine formulation can be conveniently modified to accommodateantigenic drift in circulating virus. Typically the modification will bein the G and/or F proteins. An entire G or F gene, or a genome segmentencoding a particular immunogenic region thereof, from one RSV strain isincorporated into a gene position-shifted RSV genome or antigenome cDNAby replacement of a corresponding region in a recipient clone of adifferent RSV strain or subgroup, or by adding one or more copies of thegene, such that several antigenic forms are represented. Progeny virusproduced from the modified RSV clone can then be used in vaccinationprotocols against emerging RSV strains.

[0156] A variety of additional embodiments of the invention involve theaddition or substitution of only a portion of a donor gene of interestto the recipient gene position-shifted RSV genome or antigenome.Commonly, non-coding nucleotides such as cis-acting regulatory elementsand intergenic sequences need not be transferred with the donor genecoding region. Thus, a coding sequence (e.g., a partial or complete openreading frame (ORF)) of a particular gene may be added or substituted tothe partial or complete background genome or antigenome under control ofa heterologous promoter (e.g., a promoter existing in the backgroundgenome or antigenome) of a counterpart gene or different gene ascompared to the donor sequence. A variety of additional genome segmentsprovide useful donor polynucleotides for inclusion within a chimericgenome or antigenome to express gene position-shifted RSV having noveland useful properties. For example, heterologous genome segments mayencode part or all of a glycoprotein cytoplasmic tail region,transmembrane domain or ectodomain, an epitopic site or region, abinding site or region containing a binding site, an active site orregion containing an active site, etc., of a selected protein from ahuman or bovine RSV. These and other genome segments can be added to acomplete background genome or antigenome or substituted therein for acounterpart genome segment to yield novel chimeric RSV recombinants.Certain recombinants will express a chimeric protein, e.g., a proteinhaving a cytoplasmic tail and/or transmembrane domain of one RSV fusedto an ectodomain of another RSV.

[0157] Genes and genome segments for use within the geneposition-shifted RSV of the invention embrace an assemblage of alternatepolynucleotides having a range of size and sequence variation. Usefulgenome segments in this regard range from about 15-35 nucleotides in thecase of genome segments encoding small functional domains of proteins,e.g., epitopic sites, to about 50, 75, 100, 200-500, and 500-1,500 ormore nucleotides for genome segments encoding larger domains or proteinregions. Selection of counterpart genes and genome segments relies onsequence identity or linear correspondence in the genome between thesubject counterparts. In this context, a selected human or bovinepolynucleotide “reference sequence” is defined as a sequence or portionthereof present in either the donor or recipient genome or antigenome.This reference sequence is used as a defined sequence to provide arationale basis for sequence comparison with the counterpartheterologous sequence. For example, the reference sequence may be adefined a segment of a CDNA or gene, or a complete cDNA or genesequence. Generally, a reference sequence for use in definingcounterpart genes and genome segments is at least 20 nucleotides inlength, frequently at least 25 nucleotides in length, and often at least50 nucleotides in length. Since two polynucleotides may each (1)comprise a sequence (i.e., a portion of the complete polynucleotidesequence) that is similar between the two polynucleotides, and (2) mayfurther comprise a sequence that is divergent between-the twopolynucleotides, sequence comparisons between two (or more)polynucleotides are typically performed by comparing sequences of thetwo polynucleotides over a “comparison window” to identify and comparelocal regions of sequence similarity. A “comparison window”, as usedherein, refers to a conceptual segment of at least 20 contiguousnucleotide positions wherein a polynucleotide sequence may be comparedto a reference sequence of at least 20 contiguous nucleotides andwherein the portion of the polynucleotide sequence in the comparisonwindow may comprise additions or deletions (i.e., gaps) of 20 percent orless as compared to the reference sequence (which does not compriseadditions or deletions) for optimal alignment of the two sequences.Optimal alignment of sequences for aligning a comparison window may beconducted by the local homology algorithm of (Smith & Waterman, Adv.Appl. Math. 2:482, 1981), by the homology alignment algorithm of(Needleman & Wunsch, J. Mol. Biol. 48:443, 1970), by the search forsimilarity method of (Pearson & Lipman, Proc. Natl. Acad. Sci. USA85:2444, 1988) (each of which is incorporated by reference), bycomputerized implementations of these algorithms (GAP, BESTFIT, FASTA,and TFASTA in the Wisconsin Genetics Software Package Release 7.0,Genetics Computer Group, 575 Science Dr., Madison, Wis., incorporatedherein by reference), or by inspection, and the best alignment (i.e.,resulting in the highest percentage of sequence similarity over thecomparison window) generated by the various methods is selected. Theterm “sequence identity” means that two polynucleotide sequences areidentical (i.e., on a nucleotide-by-nucleotide basis) over the window ofcomparison. The term “percentage of sequence identity” is calculated bycomparing two optimally aligned sequences over the window of comparison,determining the number of positions at which the identical nucleic acidbase (e.g., A, T, C, G, U, or I) occurs in both sequences to yield thenumber of matched positions, dividing the number of matched positions bythe total number of positions in the window of comparison (i.e., thewindow size), and multiplying the result by 100 to yield the percentageof sequence identity.

[0158] Corresponding residue positions, e.g., two different human RSVsor between a bovine and human RSV, may be divergent, identical or maydiffer by conservative amino acid substitutions. Conservative amino acidsubstitutions refer to the interchangeability of residues having similarside chains. For example, a conservative group of amino acids havingaliphatic side chains is glycine, alanine, valine, leucine, andisoleucine; a group of amino acids having aliphatic-hydroxyl side chainsis serine and threonine; a group of amino acids having amide-containingside chains is asparagine and glutamine; a group of amino acids havingaromatic side chains is phenylalanine, tyrosine, and tryptophan; a groupof amino acids having basic side chains is lysine, arginine, andhistidine; and a group of amino acids having sulfur-containing sidechains is cysteine and methionine. Preferred conservative amino acidssubstitution groups are: valine-leucine-isoleucine,phenylalanine-tyrosine, lysine-arginine, alanine-valine, andasparagine-glutamine. Stereoisomers (e.g., D-amino acids) of the twentyconventional amino acids, unnatural amino acids such asα,α-disubstituted amino acids, N-alkyl amino acids, lactic acid, andother unconventional amino acids may also be suitable components forpolypeptides of the present invention. Examples of unconventional aminoacids include: 4-hydroxyproline, γ-carboxyglutamate,ε-N,N,N-trimethyllysine, ε-N-acetyllysine, O-phosphoserine,N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine,ω-N-methylarginine, and other amino and imino acids (e.g.,4-hydroxyproline). Moreover, amino acids may be modified byglycosylation, phosphorylation and the like.

[0159] The present invention employs cDNA-based methods to construct avariety of recombinant, gene position-shifted RSV viruses and subviralparticles. These recombinant RSV offer improved characteristics ofattenuation and immunogenicity for use as vaccine agents. Desiredphenotypic changes that are engineered into gene position-shifted RSVinclude, but are not limited to, attenuation in culture or in a selectedhost environment, resistance to reversion from the attenuated phenotype,enhanced immunogenic characteristics (e.g., as determined byenhancement, or diminution, of an elicited immune response),upregulation or downregulation of transcription and/or translation ofselected viral products, etc. In preferred aspects of the invention,attenuated, gene position-shifted RSV are produced in which the chimericgenome or antigenome is further modified by introducing one or moreattenuating mutations specifying an attenuating phenotype. Thesemutations may be generated de novo and tested for attenuating effectsaccording to a rational design mutagenesis strategy as described in theabove-incorporated references. Alternatively, the attenuating mutationscan be identified in a biologically derived mutant RSV and thereafterincorporated into the gene position-shifted RSV of the invention.

[0160] Attenuating mutations in biologically derived RSV forincorporation within a gene position-shifted RSV vaccine strain mayoccur naturally or may be introduced into wild-type RSV strains by wellknown mutagenesis procedures. For example, incompletely attenuatedparental RSV strains can be produced by chemical mutagenesis duringvirus growth in cell cultures to which a chemical mutagen has beenadded, by selection of virus that has been subjected to passage atsuboptimal temperatures in order to introduce growth restrictionmutations, or by selection of a mutagenized virus that produces smallplaques (sp) in cell culture, as generally described herein and in U.S.Ser. No. U.S. Pat. No. 5,922,326, issued Jul. 13, 1999, incorporatedherein by reference.

[0161] By “biologically derived RSV” is meant any RSV not produced byrecombinant means. Thus, biologically derived RSV include naturallyoccurring RSV of all subgroups and strains, including, e.g., naturallyoccurring RSV having a wild-type genomic sequence and RSV having genomicvariations from a reference wild-type RSV sequence, e.g., RSV having amutation specifying an attenuated phenotype. Likewise, biologicallyderived RSV include RSV mutants derived from a parental RSV strain by,inter alia, artificial mutagenesis and selection procedures.

[0162] To produce a satisfactorily attenuated RSV from biologicallyderived strains, mutations are preferably introduced into a parentalstrain which has been incompletely or partially attenuated, such as thewell known ts-1 or ts-1NG or cpRSV mutants of the A2 strain of RSVsubgroup A, or derivatives or subclones thereof. Using these and otherpartially attenuated strains additional mutation(s) can be generatedthat further attenuate the strain, e.g., to a desired level ofrestricted replication in a mammalian host, while retaining sufficientimmunogenicity to confer protection in vaccinees.

[0163] Partially attenuated mutants of the subgroup A or B virus can beproduced by well known methods of biologically cloning wild-type virusin an acceptable cell substrate and developing, e.g., cold-passagedmutants thereof, subjecting the virus to chemical mutagenesis to producets mutants, or selecting small plaque or similar phenotypic mutants(see, e.g., Murphy et al., International Publication WO 93/21310,incorporated herein by reference). For virus of subgroup B, anexemplary, partially attenuated parental virus is cp 23, which is amutant of the B1 strain of subgroup B.

[0164] Various known selection techniques may be combined to producepartially attenuated mutants from non-attenuated subgroup A or B strainswhich are useful for further derivatization as described herein.Further, mutations specifying attenuated phenotypes may be introducedindividually or in combination in incompletely attenuated subgroup A orB virus to produce vaccine virus having multiple, defined attenuatingmutations that confer a desired level of attenuation and immunogenicityin vaccinees.

[0165] As noted above, production of a sufficiently attenuatedbiologically derived RSV mutant can be accomplished by several knownmethods. One such procedure involves subjecting a partially attenuatedvirus to passage in cell culture at progressively lower, attenuatingtemperatures. For example, whereas wild-type virus is typicallycultivated at about 34-37° C., the partially attenuated mutants areproduced by passage in cell cultures (e.g., primary bovine kidney cells)at suboptimal temperatures, e.g., 20-26° C. Thus, the cp mutant or otherpartially attenuated strain, e.g., ts-1 or spRSV, is adapted toefficient growth at a lower temperature by passage in MRC-5 or Verocells, down to a temperature of about 20-24° C., preferably 20-22° C.This selection of mutant RSV during cold-passage substantially reducesany residual virulence in the derivative strains as compared to thepartially attenuated parent.

[0166] Alternatively, specific mutations can be introduced intobiologically derived RSV by subjecting a partially attenuated parentvirus to chemical mutagenesis, e.g., to introduce ts mutations or, inthe case of viruses which are already ts, additional ts mutationssufficient to confer increased attenuation and/or stability of the tsphenotype of the attenuated derivative. Means for the introduction of tsmutations into RSV include replication of the virus in the presence of amutagen such as 5-fluorouridine or 5-fluorouracil in a concentration ofabout 10⁻³ to 10⁻⁵ M, preferably about 10⁻⁴ M, exposure of virus tonitrosoguanidine at a concentration of about 100 μg/ml, according to thegeneral procedure described in, e.g., (Gharpure et al., J. Virol.3:414-421, 1969 and Richardson et al., J. Med. Virol. 3:91-100, 1978),or genetic introduction of specific ts mutations. Other chemicalmutagens can also be used. Attenuation can result from a ts mutation inalmost any RSV gene, although a particularly amenable target for thispurpose has been found to be the polymerase (L) gene.

[0167] The level of temperature sensitivity of replication in exemplaryattenuated RSV for use within the invention is determined by comparingits replication at a permissive temperature with that at severalrestrictive temperatures. The lowest temperature at which thereplication of the virus is reduced 100-fold or more in comparison withits replication at the permissive temperature is termed the shutofftemperature. In experimental animals and humans, both the replicationand virulence of RSV correlate with the mutant's shutoff temperature.Replication of mutants with a shutoff temperature of 39° C. ismoderately restricted, whereas mutants with a shutoff of 38° C.replicate less well and symptoms of illness are mainly restricted to theupper respiratory tract. A virus with a shutoff temperature of 35° C. to37° C. will typically be fully attenuated in chimpanzees andsubstantially attenuated in humans. Thus, attenuated biologicallyderived mutant and gene position-shifted RSV of the invention which arets will have a shutoff temperature in the range of about 35° C. to 39°C., and preferably from 35° C. to 38° C. The addition of a ts mutationinto a partially attenuated strain produces a multiply attenuated virususeful within vaccine compositions of the invention.

[0168] A number of attenuated RSV strains as candidate vaccines forintranasal administration have been developed using multiple rounds ofchemical mutagenesis to introduce multiple mutations into a virus whichhad already been attenuated during cold-passage (e.g., Connors et al.,Virology 208: 478-484, 1995; Crowe et al., Vaccine 12: 691-699, 1994;and Crowe et al., Vaccine 12: 783-790, 1994, incorporated herein byreference). Evaluation in rodents, chimpanzees, adults and infantsindicate that certain of these candidate vaccine strains are relativelystable genetically, are highly immunogenic, and may be satisfactorilyattenuated. Nucleotide sequence analysis of some of these attenuatedviruses indicates that each level of increased attenuation is associatedwith specific nucleotide and amino acid substitutions. Theabove-incorporated references also disclose how to routinely distinguishbetween silent incidental mutations and those responsible for phenotypedifferences by introducing the mutations, separately and in variouscombinations, into the genome or antigenome of infectious RSV clones.This process coupled with evaluation of phenotype characteristics ofparental and derivative virus identifies mutations responsible for suchdesired characteristics as attenuation, temperature sensitivity,cold-adaptation, small plaque size, host range restriction, etc.

[0169] Mutations thus identified are compiled into a “menu” and are thenintroduced as desired, singly or in combination, to calibrate a geneposition-shifted RSV vaccine virus to an appropriate level ofattenuation, immunogenicity, genetic resistance to reversion from anattenuated phenotype, etc., as desired. Preferably, the chimeric RSV ofthe invention are attenuated by incorporation of at least one, and morepreferably two or more, attenuating mutations identified from such amenu, which may be defmed as a group of known mutations within a panelof biologically derived mutant RSV strains. Preferred panels of mutantRSV strains described herein are cold passaged (cp) and/or temperaturesensitive (ts) mutants, for example a panel comprised of RSV mutantsdesignated cpts RSV 248 (ATCC VR 2450), cpts RSV 248/404 (ATCC VR 2454),cpts RSV 248/955 (ATCC VR 2453), cpts RSV 530 (ATCC VR 2452), cpts RSV530/1009 (ATCC VR 2451), cpts RSV 530/1030 (ATCC VR 2455), RSV B-1cp52/2B5 (ATCC VR 2542), and RSV B-1 cp-23 (ATCC VR 2579) (eachdeposited under the terms of the Budapest Treaty with the American TypeCulture Collection (ATCC) of 10801 University Boulevard, Manassas, Va.20110-2209, U.S.A., and granted the above identified accession numbers).

[0170] From this exemplary panel of biologically derived mutants, alarge menu of attenuating mutations are provided which can each becombined with any other mutation(s) within the panel for calibrating thelevel of attenuation in a recombinant, gene position-shifted RSV forvaccine use. Additional mutations may be derived from RSV having non-tsand non-cp attenuating mutations as identified, e.g., in small plaque(sp), cold-adapted (ca) or host-range restricted (hr) mutant strains.Attenuating mutations may be selected in coding portions of a donor orrecipient RSV gene or in non-coding regions such as a cis-regulatorysequence. For example, attenuating mutations may include single ormultiple base changes in a gene start sequence, as exemplified by asingle or multiple base substitution in the M2 gene start sequence atnucleotide 7605.

[0171] Gene position-shifted RSV designed and selected for vaccine useoften have at least two and sometimes three or more attenuatingmutations to achieve a satisfactory level of attenuation for broadclinical use. In one embodiment, at least one attenuating mutationoccurs in the RSV polymerase gene (either in the donor or recipientgene) and involves a nucleotide substitution specifying an amino acidchange in the polymerase protein specifying a temperature-sensitive (ts)phenotype. Exemplary gene position-shifted RSV in this contextincorporate one or more nucleotide substitutions in the large polymerasegene L resulting in an amino acid change at amino acid Asn43, Phe521,Gln831, Met1169, or Tyr1321, as exemplified by the changes, Leu forPhe521, Leu for Gln831, Val for Met1169, and Asn for Tyr1321.Alternately or additionally, gene position-shifted RSV of the inventionmay incorporate a ts mutation in a different RSV gene, e.g., in the M2gene. Preferably, two or more nucleotide changes are incorporated in acodon specifying an attenuating mutation, e.g., in a codon specifying ats mutation, thereby decreasing the likelihood of reversion from anattenuated phenotype.

[0172] In accordance with the methods of the invention, geneposition-shifted RSV can be readily constructed and characterized thatincorporate at least one and up to a full complement of attenuatingmutations present within a panel of biologically derived mutant RSVstrains. Thus, mutations can be assembled in any combination from aselected panel of mutants, for example, cpts RSV 248 (ATCC VR 2450),cpts RSV 248/404 (ATCC VR 2454), cpts RSV 248/955 (ATCC VR 2453), cptsRSV 530 (ATCC VR 2452), cpts RSV 530/1009 (ATCC VR 2451), cpts RSV530/1030 (ATCC VR 2455), RSV B-1 cp52/2B5 (ATCC VR 2542), and RSV B-1cp-23 (ATCC VR 2579). In this manner, attenuation of recombinant vaccinecandidates can be finely calibrated for use in one or more classes ofpatients, including seronegative infants.

[0173] In more specific embodiments, gene position-shifted RSV forvaccine use incorporate at least one and up to a full complement ofattenuating mutations specifying a temperature-sensitive and/orattenuating amino acid substitution at Asn43, Phe521, Gln831, Met1169 orTyr1321 in the RSV polymerase gene L, or a temperature- sensitivenucleotide substitution in the gene-start sequence of gene M2.Alternatively or additionally, gene position-shifted RSV of claim mayincorporate at least one and up to a full complement of mutations fromcold-passaged attenuated RSV, for example one or more mutationsspecifying an amino acid substitution at Val267 in the RSV N gene,Glu218 or Thr523 in the RSV F gene, Cys319 or His1690 in the RSVpolymerase gene L.

[0174] In other detailed embodiments, the gene position-shifted RSV ofthe invention is further modified to incorporate attenuating mutationsselected from (i) a panel of mutations specifying temperature-sensitiveamino acid substitutions Gln831 to Leu, and Tyr1321 to Asn in the RSVpolymerase gene L; (ii) a temperature-sensitive nucleotide substitutionin the gene-start sequence of gene M2; (iii) an attenuating panel ofmutations adopted from cold-passaged RSV specifying amino acidsubstitutions Va1267 Ile in the RSV N gene, and Cys319 Tyr and His1690Tyr in the RSV polymerase gene L; or (iv) deletion or ablation ofexpression of one or more of the RSV SH, NS1, NS2, G and M2-2 genes.Preferably, these and other examples of gene position-shifted RSVincorporate at least two attenuating mutations adopted from biologicallyderived mutant RSV, which may be derived from the same or differentbiologically derived mutant RSV strains. Also preferably, theseexemplary mutants have one or more of their attenuating mutationsstabilized by multiple nucleotide changes in a codon specifying themutation.

[0175] In accordance with the foregoing description, the ability toproduce infectious RSV from cDNA permits introduction of specificengineered changes within gene position-shifted RSV. In particular,infectious, recombinant RSV are employed for identification of specificmutation(s) in biologically derived, attenuated RSV strains, for examplemutations which specify ts, ca, att and other phenotypes. Desiredmutations are thus identified and introduced into recombinant, geneposition-shifted RSV vaccine strains. The capability of producing virusfrom cDNA allows for routine incorporation of these mutations,individually or in various selected combinations, into a full-lengthcDNA clone, whereafter the phenotypes of rescued recombinant virusescontaining the introduced mutations to be readily determined.

[0176] By identifying and incorporating specific, biologically derivedmutations associated with desired phenotypes, e.g., a cp or tsphenotype, into infectious chimeric RSV clones, the invention providesfor other, site-specific modifications at, or within close proximity to,the identified mutation. Whereas most attenuating mutations produced inbiologically derived RSV are single nucleotide changes, other “sitespecific” mutations can also be incorporated by recombinant techniquesinto biologically derived or recombinant RSV. As used herein,site-specific mutations include insertions, substitutions, deletions orrearrangements of from 1 to 3, up to about 5-15 or more alterednucleotides (e.g., altered from a wild-type RSV sequence, from asequence of a selected mutant RSV strain, or from a parent recombinantRSV clone subjected to mutagenesis). Such site-specific mutations may beincorporated at, or within the region of, a selected, biologicallyderived mutation. Alternatively, the mutations can be introduced invarious other contexts within an RSV clone, for example at or near acis-acting regulatory sequence or nucleotide sequence encoding a proteinactive site, binding site, immunogenic epitope, etc. Site-specific RSVmutants typically retain a desired attenuating phenotype, but mayadditionally exhibit altered phenotypic characteristics unrelated toattenuation, e.g., enhanced or broadened immunogenicity, and/or improvedgrowth. Further examples of desired, site-specific mutants includerecombinant RSV designed to incorporate additional, stabilizingnucleotide mutations in a codon specifying an attenuating mutation.Where possible, two or more nucleotide substitutions are introduced atcodons that specify attenuating amino acid changes in a parent mutant orrecombinant RSV clone, yielding a biologically derived or recombinantRSV having genetic resistance to reversion from an attenuated phenotype.In other embodiments, site-specific nucleotide substitutions, additions,deletions or rearrangements are introduced upstream (N-terminaldirection) or downstream (C-terminal direction), e.g., from 1 to 3, 5-10and up to 15 nucleotides or more 5′ or 3′, relative to a targetednucleotide position, e.g., to construct or ablate an existing cis-actingregulatory element.

[0177] In addition to single and multiple point mutations andsite-specific mutations, changes to gene position-shifted RSV disclosedherein include deletions, insertions, substitutions or rearrangements ofwhole genes or genome segments. These mutations may alter small numbersof bases (e.g., from 15-30 bases, up to 35-50 bases or more), largeblocks of nucleotides (e.g., 50-100, 100-300, 300-500, 500-1,000 bases),or nearly complete or complete genes (e.g., 1,000-1,500 nucleotides,1,500-2,500 nucleotides, 2,500-5,000, nucleotides, 5,00-6,5000nucleotides or more) in the donor or recipient genome or antigenome,depending upon the nature of the change (i.e., a small number of basesmay be changed to insert or ablate an immunogenic epitope or change asmall genome segment, whereas large block(s) of bases are involved whengenes or large genome segments are added, substituted, deleted orrearranged.

[0178] In alternative aspects of the invention, the infectious geneposition-shifted RSV produced from a cDNA-expressed genome or antigenomecan be any of the RSV or RSV-like strains, e.g., human, bovine, murine,etc., or of any pneumovirus, e.g., pneumonia virus of mice avianpneumovirus (previously called turkey rhinotracheitis virus). Toengender a protective immune response, the RSV strain may be one whichis endogenous to the subject being immunized, such as human RSV beingused to immunize humans. The genome or antigenome of endogenous RSV canbe modified, however, to express RSV genes or genome segments from acombination of different sources, e.g., a combination of genes or genomesegments from different RSV species, subgroups, or strains, or from anRSV and another respiratory pathogen such as PIV.

[0179] Introduction of the foregoing defined mutations into aninfectious, gene position-shifted RSV clone can be achieved by a varietyof well known methods. By “infectious clone” with regard to DNA is meantcDNA or its product, synthetic or otherwise, which can be transcribedinto genomic or antigenomic RNA capable of serving as template toproduce the genome of an infectious virus or subviral particle. Thus,defined mutations can be introduced by conventional techniques (e.g.,site-directed mutagenesis) into a cDNA copy of the genome or antigenome.The use of antigenome or genome cDNA subfragments to assemble a completeantigenome or genome cDNA as described herein has the advantage thateach region can be manipulated separately (smaller cDNAs are easier tomanipulate than large ones) and then readily assembled into a completecDNA. Thus, the complete antigenome or genome cDNA, or any subfragmentthereof, can be used as template for oligonucleotide-directedmutagenesis. This can be through the intermediate of a single-strandedphagemid form, such as using the Muta-gene® kit of Bio-Rad Laboratories(Richmond, Calif.) or a method using a double-stranded plasmid directlyas template such as the Chameleon mutagenesis kit of Stratagene (LaJolla, Calif.), or by the polymerase chain reaction employing either anoligonucleotide primer or template which contains the mutation(s) ofinterest. A mutated subfragment can then be assembled into the completeantigenome or genome cDNA. A variety of other mutagenesis techniques areknown and available for use in producing the mutations of interest inthe RSV antigenome or genome cDNA. Mutations can vary from singlenucleotide changes to replacement of large cDNA pieces containing one ormore genes or genome regions.

[0180] Thus, in one illustrative embodiment mutations are introduced byusing the Muta-gene phagemid in vitro mutagenesis kit available fromBio-Rad. In brief, cDNA encoding a portion of an RSV genome orantigenome is cloned into the plasmid pTZ 18U, and used to transformCJ236 cells (Life Technologies). Phagemid preparations are prepared asrecommended by the manufacturer. Oligonucleotides are designed formutagenesis by introduction of an altered nucleotide at the desiredposition of the genome or antigenome. The plasmid containing thegenetically altered genome or antigenome fragment is then amplified andthe mutated piece is then reintroduced into the full-length genome orantigenome clone.

[0181] The invention also provides methods for producing an infectiousgene position-shifted RSV from one or more isolated polynucleotides,e.g., one or more cDNAs. According to the present invention cDNAencoding a RSV genome or antigenome is constructed for intracellular orin vitro coexpression with the necessary viral proteins to forminfectious RSV. By “RSV antigenome” is meant an isolated positive-sensepolynucleotide molecule which serves as the template for the synthesisof progeny RSV genome. Preferably a cDNA is constructed which is apositive-sense version of the RSV genome, corresponding to thereplicative intermediate RNA, or antigenome, so as to minimize thepossibility of hybridizing with positive-sense transcripts of thecomplementing sequences that encode proteins necessary to generate atranscribing, replicating nucleocapsid, i.e., sequences that encode N,P, L and M2(ORF1) protein. In an RSV minigenome system, genome andantigenome were equally active in rescue, whether complemented by RSV orby plasmids, indicating that either genome or antigenome can be used andthus the choice can be made on methodologic or other grounds.

[0182] A native RSV genome typically comprises a negative-sensepolynucleotide molecule which, through complementary viral mRNAs,encodes eleven species of viral proteins, i.e., the nonstructuralspecies NS1 and NS2, N, P, matrix (M), small hydrophobic (SH),glycoprotein (G), fusion (F), M2(ORF1), M2(ORF2), and L, substantiallyas described in (Mink et al., Virology 185:615-624, 1991; Stec et al.,Virology 183:273-287, 1991; and Connors et al., Virol. 208:478-484,1995; Collins et al., Proc. Nat. Acad. Sci. USA 93:81-85, 1996), eachincorporated herein by reference. For purposes of the present inventionthe genome or antigenome of the recombinant RSV of the invention needonly contain those genes or portions thereof necessary to render theviral or subviral particles encoded thereby infectious. Further, thegenes or portions thereof may be provided by more than onepolynucleotide molecule, i.e., a gene may be provided by complementationor the like from a separate nucleotide molecule, or can be expresseddirectly from the genome or antigenome cDNA.

[0183] By recombinant RSV is meant a RSV or RSV-like viral or subviralparticle derived directly or indirectly from a recombinant expressionsystem or propagated from virus or subviral particles producedtherefrom. The recombinant expression system will employ a recombinantexpression vector which comprises an operably linked transcriptionalunit comprising an assembly of at least a genetic element or elementshaving a regulatory role in RSV gene expression, for example, apromoter, a structural or coding sequence which is transcribed into RSVRNA, and appropriate transcription initiation and termination sequences.

[0184] To produce infectious RSV from cDNA-expressed genome orantigenome, the genome or antigenome is coexpressed with those RSVproteins necessary to (i) produce a nucleocapsid capable of RNAreplication, and (ii) render progeny nucleocapsids competent for bothRNA replication and transcription. Transcription by the genomenucleocapsid provides the other RSV proteins and initiates a productiveinfection. Alternatively, additional RSV proteins needed for aproductive infection can be supplied by coexpression.

[0185] An RSV antigenome may be constructed for use in the presentinvention by assembling cloned cDNA segments, representing in aggregatethe complete antigenome, by polymerase chain reaction (PCR; describedin, e.g., U.S. Pat. Nos. 4,683,195 and 4,683,202, and PCR Protocols: AGuide to Methods and Applications, Innis et al., eds., Academic Press,San Diego, 1990, incorporated herein by reference) ofreverse-transcribed copies of RSV mRNA or genome RNA. For example, cDNAscontaining the lefthand end of the antigenome, spanning from anappropriate promoter (e.g., T7 RNA polymerase promoter) and the leaderregion complement to the SH gene, are assembled in an appropriateexpression vector, such as a plasmid (e.g., pBR322) or various availablecosmid, phage, or DNA virus vectors. The vector may be modified bymutagenesis and/or insertion of synthetic polylinker containing uniquerestriction sites designed to facilitate assembly. For example, aplasmid vector described herein was derived from pBR322 by replacementof the PstI-EcoR1 fragment with a synthetic DNA containing convenientrestriction enzyme sites. Use of pBR322 as a vector stabilizednucleotides 3716-3732 of the RSV sequence, which otherwise sustainednucleotide deletions or insertions, and propagation of the plasmid wasin bacterial strain DH10B to avoid an artifactual duplication andinsertion which otherwise occurred in the vicinity of nt 4499. For easeof preparation the G, F and M2 genes can be assembled in a separatevector, as can be the L and trailer sequences. The right-hand end (e.g.,L and trailer sequences) of the antigenome plasmid may containadditional sequences as desired, such as a flanking ribozyme and tandemT7 transcriptional terminators. The ribozyme can be hammerhead type(e.g., Grosfeld et al., J. Virol. 69:5677-5686, 1995), which would yielda 3′ end containing a single nonviral nucleotide, or can any of theother suitable ribozymes such as that of hepatitis delta virus (Perrottaet al., Nature 350:434-436, 1991) which would yield a 3′ end free ofnon-RSV nucleotides. A middle segment (e.g., G-to-M2 piece) is insertedinto an appropriate restriction site of the leader-to-SH plasmid, whichin turn is the recipient for the L-trailer-ribozyme-terminator piece,yielding a complete antigenome. In an illustrative example describedherein, the leader end was constructed to abut the promoter for T7 RNApolymerase which included three transcribed G residues for optimalactivity; transcription donates these three nonviral G's to the 5′ endof the antigenome. These three nonviral G residues can be omitted toyield a 5′ end free of nonviral nucleotides. To generate a nearlycorrect 3′ end, the trailer end was constructed to be adjacent to ahammerhead ribozyme, which upon cleavage would donate a single3′-phosphorylated U residue to the 3′ end of the encoded RNA.

[0186] In certain embodiments of the invention, complementing sequencesencoding proteins necessary to generate a transcribing, replicating RSVnucleocapsid are provided by one or more helper viruses. Such helperviruses can be wild-type or mutant. Preferably, the helper virus can bedistinguished phenotypically from the virus encoded by the RSV cDNA. Forexample, it is desirable to provide monoclonal antibodies which reactimmunologically with the helper virus but not the virus encoded by theRSV cDNA. Such antibodies can be neutralizing antibodies. In someembodiments, the antibodies can be used to neutralize the helper virusbackground to facilitate identification and recovery of the recombinantvirus, or in affinity chromatography to separate the helper virus fromthe recombinant virus. Mutations can be introduced into the RSV cDNAwhich render the recombinant RSV nonreactive or resistant toneutralization with such antibodies.

[0187] A variety of nucleotide insertions and deletions can be made inthe gene position-shifted RSV genome or antigenome to generate aproperly attenuated clone. The nucleotide length of the genome ofwild-type human RSV (15,222 nucleotides) is a multiple of six, andmembers of the Paramyxovirus and Morbillivirus genera typically abide bya “rule of six,” i.e., genomes (or minigenomes) replicate efficientlyonly when their nucleotide length is a multiple of six (thought to be arequirement for precise spacing of nucleotide residues relative toencapsidating NP protein). Alteration of RSV genome length by singleresidue increments had no effect on the efficiency of replication, andsequence analysis of several different minigenome mutants followingpassage showed that the length differences were maintained withoutcompensatory changes. Thus, RSV lacks the strict requirement of genomelength being a multiple of six, and nucleotide insertions and deletionscan be made in the RSV genome or antigenome without defeatingreplication of the recombinant RSV of the present invention.

[0188] Alternative means to construct cDNA encoding a geneposition-shifted RSV genome or antigenome include by reversetranscription-PCR using improved PCR conditions (e.g., as described inCheng et al., Proc. Natl. Acad. Sci. USA 91:5695-5699, 1994; Samal etal., J. Virol. 70:5075-5082, 1996, each incorporated herein byreference) to reduce the number of subunit cDNA components to as few asone or two pieces. In other embodiments different promoters can be used(e.g., T3, SP6) or different ribozymes (e.g., that of hepatitis deltavirus. Different DNA vectors (e.g., cosmids) can be used for propagationto better accommodate the large size genome or antigenome.

[0189] The N, P and L proteins, necessary for RNA replication, requirean RNA polymerase elongation factor such as the M2(ORF1) protein forprocessive transcription. Thus M2(ORF1) or a substantially equivalenttranscription elongation factor for negative strand RNA viruses isrequired for the production of infectious RSV and is a necessarycomponent of functional nucleocapsids during productive infection.

[0190] The need for the M2(ORF1) protein is consistent with its role asa transcription elongation factor. The need for expression of the RNApolymerase elongation factor protein for negative strand RNA viruses isa feature of the present invention. M2(ORF1) can be supplied byexpression of the complete M2-gene, either by the chimeric genome orantigenome or by coexpression therewith, although in this form thesecond ORF2 may also be expressed and have an inhibitory effect on RNAreplication. Therefore, for production of infectious virus using thecomplete M2 gene the activities of the two ORFs should be balanced topermit sufficient expression of M2(ORFl) to provide transcriptionelongation activity yet not so much of M2(ORF2) to inhibit RNAreplication. Alternatively, the ORF1 protein is provided from a cDNAengineered to lack ORF2 or which encodes a defective ORF2. Efficiency ofvirus production may also be improved by co-expression of additionalviral protein genes, such as those encoding envelope constituents (i.e.,SH, M, G, F proteins).

[0191] In accordance with these results concerning M2(ORF2), anotherexemplary embodiment of the invention is provided comprising a geneposition-shifted RSV that incorporate a mutation of M2(ORF2) (Collinsand Wertz, J. Virol. 54:65-71, 1985; Collins et al., J. Gen. Virol.71:3015-3020, 1990, Collins et al., Proc. Natl. Acad. Sci. USA 93:81-85,1996, each incorporated herein by reference) to yield novel RSV vaccinecandidates (see U.S. Provisional Patent Application No. 60/143,097,filed by Collins et al. on Jul. 9, 1999, incorporated herein byreference). In certain aspects, expression of M2 ORF2 is reduced orablated by modifying the recombinant RSV genome or antigenome toincorporate a frame shift mutation or one or stop codons in M2 ORF2yielding a “knock out” viral clone. Alternatively, M2 ORF2 is deleted inwhole or in part to render the M2-2 protein partially or entirelynon-functional or to disrupt its expression altogether to yield a M2ORF2 “deletion mutant” chimeric RSV. Alternatively, the M2-2 ORF may betranspositioned in the genome or antigenome to a more promoter-proximalor promoter-distal position compared to the natural gene order positionof M2-2 gene to up-regulate or down-regulate expression of the M2-2 ORF.In additional embodiments, the M2-2 ORF is incorporated in the genome orantigenome as a separate gene having a gene start and gene end gene endsignal, which modification results in up-regulation of the M2-2 ORF.

[0192] The gene position-shifted RSV of the invention that incorporatemutations in M2 ORF2 possess highly desirable phenotypic characteristicsfor vaccine development. The above identified modifications in therecombinant genome or antigenome specify one or more desired phenotypicchanges in the resulting virus or subviral particle. Vaccine candidatesare thus generated that exhibit one or more characteristics identifiedas (i) a change in MRNA transcription, (ii) a change in the level ofviral protein expression; (iii) a change in genomic or antigenomic RNAreplication, (iv) a change in viral growth characteristics, (v), achange in viral plaque size, and/or (vi) a change in cytopathogenicity.

[0193] In exemplary RSV recombinants incorporating an M2 ORF 2 deletionor knock out mutation, desired phenotypic changes include attenuation ofviral growth compared to growth of a corresponding wild-type or mutantparental RSV strain. In more detailed aspects, viral growth in cellculture may be attenuated by approximately 10-fold or more attributableto mutations in M2 ORF2. Kinetics of viral growth are also shown to bemodified in a manner that is beneficial for vaccine development.

[0194] Also included within the invention are M2-ORF 2 deletion andknock out mutant RSV that exhibit delayed kinetics of viral MRNAsynthesis compared to kinetics of mRNA synthesis of correspondingwild-type or mutant parental RSV strains. Despite these delayedtranscription kinetics, these novel vaccine candidates exhibit anincrease in cumulative mRNA synthesis compared to parental virus. Thesephenotypic changes typically are associated with an increase in viralprotein accumulation in infected cells compared to protein accumulationin cells infected with wild-type or other parental RSV strains. At thesame time, viral RNA replication is reduced in M2 ORF2 geneposition-shifted RSV compared to that of a parental RSV strain havingnormal M2 ORF2 function, whereby accumulation of genomic or antigenomicRNA is reduced.

[0195] Within preferred aspects of the invention, chimeric M2 ORF2deletion and “knock out” RSV are engineered to express undiminished or,more typically, increased levels of viral antigen(s) while alsoexhibiting an attenuated phenotype. Immunogenic potential is thuspreserved due to the undiminished or increased mRNA transcription andantigen expression, while attenuation is achieved through incorporationof the heterologous gene(s) or gene segment(s) and concomitantreductions in RNA replication and virus growth attributable to theM2-ORF 2 deletion and knock out mutation. This novel suite of phenotypictraits is highly desired for vaccine development. Other usefulphenotypic changes that are observed in M2 ORF2 deletion and knock outgene position-shifted RSV include a large plaque phenotype and alteredcytopathogenicity compared to corresponding wild-type or mutant parentalRSV strains.

[0196] Isolated polynucleotides (e.g., cDNA) encoding a geneposition-shifted RSV genome or antigenome and, separately, or in cis, orexpressed from the antigenome or genome cDNA, the N, P, L and M2(ORF1)proteins, are inserted by transfection, electroporation, mechanicalinsertion, transduction or the like, into cells which are capable ofsupporting a productive RSV infection, e.g., HEp-2, FRhL-DBS2, MRC, andVero cells. Transfection of isolated polynucleotide sequences may beintroduced into cultured cells by, for example, calciumphosphate-mediated transfection (Wigler et al., Cell 14:725, 1978;Corsaro and Pearson, Somatic Cell Genetics 7:603, 1981; Graham and Vander Eb, Virology 52:456, 1973), electroporation (Neumann et al., EMBO J.1:841-845, 1982), DEAE-dextran mediated transfection (Ausubel et al.,(ed.) Current Protocols in Molecular Biology, John Wiley and Sons, Inc.,NY, 1987, cationic lipid-mediated transfection (Hawley-Nelson et al.,Focus 15:73-79, 1993) or a commercially available transfection regent,e.g., LipofectACE® (Life Technologies) (each of the foregoing referencesare incorporated herein by reference).

[0197] The N, P, L and M2(ORF1) proteins are encoded by one or morecDNAs and expression vectors which can be the same or separate from thatwhich encodes the genome or antigenome, and various combinationsthereof. Additional proteins may be included as desired, encoded by itsown vector or by a vector encoding a N, P, L, or M2(ORF1) protein and/orthe complete genome or antigenome. Expression of the genome orantigenome and proteins from transfected plasmids can be achieved, forexample, by each cDNA being under the control of a promoter for T7 RNApolymerase, which in turn is supplied by infection, transfection ortransduction with an expression system for the T7 RNA polymerase, e.g.,a vaccinia virus MVA strain recombinant which expresses the T7 RNApolymerase (Wyatt et al., Virology, 210:202-205, 1995, incorporatedherein by reference). The viral proteins, and/or T7 RNA polymerase, canalso be provided from transformed mammalian cells, or by transfection ofpreformed mRNA or protein.

[0198] Alternatively, synthesis of antigenome or genome can be conductedin vitro (cell-free) in a combined transcription-translation reaction,followed by transfection into cells. Or, antigenome or genome RNA can besynthesized in vitro and transfected into cells expressing RSV proteins.

[0199] To select candidate vaccine viruses according to the invention,the criteria of viability, attenuation and immunogenicity are determinedaccording to well known methods. Viruses which will be most desired invaccines of the invention must maintain viability, have a stableattenuation phenotype, exhibit replication in an immunized host (albeitat lower levels), and effectively elicit production of an immuneresponse in a vaccinee sufficient to confer protection against seriousdisease caused by subsequent infection from wild-type virus. Clearly,the heretofore known and reported RS virus mutants do not meet all ofthese criteria. Indeed, contrary to expectations based on the resultsreported for known attenuated RSV, viruses of the invention are not onlyviable and more appropriately attenuated than previous mutants, but aremore stable genetically in vivo than those previously studiedmutants—retaining the ability to stimulate a protective immune responseand in some instances to expand the protection afforded by multiplemodifications, e.g., induce protection against different viral strainsor subgroups, or protection by a different immunologic basis, e.g.,secretory versus serum immunoglobulins, cellular immunity, and the like.Prior to the invention, genetic instability of the ts phenotypefollowing replication in vivo has been common for ts viruses (Murphy etal., Infect. Immun. 37:235-242, 1982).

[0200] To propagate a gene position-shifted RSV virus for vaccine useand other purposes, a number of cell lines which allow for RSV growthmay be used. RSV grows in a variety of human and animal cells. Preferredcell lines for propagating attenuated RS virus for vaccine use includeDBS-FRhL-2, MRC-5, and Vero cells. Highest virus yields are usuallyachieved with epithelial cell lines such as Vero cells. Cells aretypically inoculated with virus at a multiplicity of infection rangingfrom about 0.001 to 1.0 or more, and are cultivated under conditionspermissive for replication of the virus, e.g., at about 30-37° C. andfor about 3-5 days, or as long as necessary for virus to reach anadequate titer. Virus is removed from cell culture and separated fromcellular components, typically by well known clarification procedures,e.g., centrifugation, and may be further purified as desired usingprocedures well known to those skilled in the art.

[0201] Gene position-shifted RSV which has been attenuated and otherwisemodified as described herein can be tested in various well known andgenerally accepted in vitro and in vivo models to confirm adequateattenuation, resistance to phenotypic reversion, and immunogenicity forvaccine use. In in vitro assays, the modified virus (e.g., a multiplyattenuated, biologically derived or recombinant RSV) is tested fortemperature sensitivity of virus replication, i.e. ts phenotype, and forthe small plaque phenotype. Modified viruses are further tested inanimal models of RSV infection. A variety of animal models have beendescribed and are summarized in (Meignier et al., eds., Animal Models ofRespiratory Syncytial Virus Infection, Merieux Foundation Publication,1991, which is incorporated herein by reference). A cotton rat model ofRSV infection is described in (U.S. Pat. No. 4,800,078 and Prince etal., Virus Res. 3:193-206, 1985), which are incorporated herein byreference, and is considered predictive of attenuation and efficacy inhumans and non-human primates. In addition, a primate model of RSVinfection using the chimpanzee is predictive of attenuation and efficacyin humans, as is described in detail in (Richardson et al., J. Med.Virol. 3:91-100, 1978; Wright et al., Infect. Immun. 37:397-400, 1982;Crowe et al., Vaccine 11:1395-1404, 1993, each incorporated herein byreference).

[0202] RSV model systems, including rodents and chimpanzees forevaluating attenuation and infectivity of RSV vaccine candidates arewidely accepted in the art and the data obtained therefrom correlatewell with RSV infection and attenuation. The mouse and cotton rat modelsare especially useful in those instances in which candidate RSV virusesdisplay inadequate growth in chimpanzees, for example in the case of RSVsubgroup B viruses.

[0203] In accordance with the foregoing description and based on theexamples below, the invention also provides isolated, infectious geneposition-shifted RSV compositions for vaccine use. The attenuatedchimeric virus which is a component of a vaccine is in an isolated andtypically purified form. By isolated is meant to refer to RSV which isin other than a native environment of a wild-type virus, such as thenasopharynx of an infected individual. More generally, isolated is meantto include the attenuated virus as a component of a cell culture orother artificial medium where it can be propagated and characterized ina controlled setting. For example, attenuated RSV of the invention maybe produced by an infected cell culture, separated from the cell cultureand added to a stabilizer.

[0204] RSV vaccines of the invention contain as an active ingredient animmunogenically effective amount of RSV produced as described herein.Biologically derived or recombinant RSV can be used directly in vaccineformulations, or lyophilized. Lyophilized virus will typically bemaintained at about 4° C. When ready for use the lyophilized virus isreconstituted in a stabilizing solution, e.g., saline or comprising SPG,Mg++ and HEPES, with or without adjuvant, as further described below.The biologically derived or recombinantly modified virus may beintroduced into a host with a physiologically acceptable carrier and/oradjuvant. Useful carriers are well known in the art, and include, e.g.,water, buffered water, 0.4% saline, 0.3% glycine, hyaluronic acid andthe like. The resulting aqueous solutions may be packaged for use as is,or lyophilized, the lyophilized preparation being combined with asterile solution prior to administration, as mentioned above. Thecompositions may contain pharmaceutically acceptable auxiliarysubstances as required to approximate physiological conditions, such aspH adjusting and buffering agents, tonicity adjusting agents, wettingagents and the like, for example, sodium acetate, sodium lactate, sodiumchloride, potassium chloride, calcium chloride, sorbitan monolaurate,triethanolamine oleate, and the like. Acceptable adjuvants includeincomplete Freund's adjuvant, aluminum phosphate, aluminum hydroxide, oralum, which are materials well known in the art. Preferred adjuvantsalso include Stimulon® QS-21 (Aquila Biopharmaceuticals, Inc.,Framingham, Mass.), MPL® (3-0-deacylated monophosphoryl lipid A; Corixa,Hamilton, Mont.), and interleukin-12 (Genetics Institute, Cambridge,Mass.).

[0205] Upon immunization with a gene position-shifted RSV vaccinecomposition as described herein, via aerosol, droplet, oral, topical orother route, the immune system of the host responds to the vaccine byproducing antibodies specific for one or more RSV virus proteins, e.g.,F and/or G glycoproteins. As a result of the vaccination the hostbecomes at least partially or completely immune to RSV infection, orresistant to developing moderate or severe RSV disease, particularly ofthe lower respiratory tract.

[0206] Gene position-shifted RSV vaccines of the invention may compriseattenuated virus that elicits an immune response against a single RSVstrain or antigenic subgroup, e.g. A or B, or against multiple RSVstrains or subgroups. In this context, the gene position-shifted RSV canelicit a monospecific immune response or a polyspecific immune responseagainst multiple RSV strains or subgroups. Alternatively, geneposition-shifted RSV having different immunogenic characteristics can becombined in a vaccine mixture or administered separately in acoordinated treatment protocol to elicit more effective protectionagainst one RSV strain, or against multiple RSV strains or subgroups.

[0207] The host to which the vaccine is administered can be any mammalsusceptible to infection by RSV or a closely related virus and capableof generating a protective immune response to antigens of thevaccinizing virus. Thus, suitable hosts include humans, non-humanprimates, bovine, equine, swine, ovine, caprine, lagamorph, rodents,etc. Accordingly, the invention provides methods for creating vaccinesfor a variety of human and veterinary uses.

[0208] The vaccine compositions containing the attenuated geneposition-shifted RSV of the invention are administered to a patientsusceptible to or otherwise at risk of RSV infection in an“immunogenically effective dose” which is sufficient to induce orenhance the individual's immune response capabilities against RSV. Inthe case of human subjects, the attenuated virus of the invention isadministered according to well established human RSV vaccine protocols,as described in, e.g., (Wright et al., Infect. Immun. 37:397-400, 1982;Kim et al., Pediatrics 52:56-63, 1973; and Wright et al., J. Pediatr.88:931-936, 1976), which are each incorporated herein by reference.Briefly, adults or children are inoculated intranasally via droplet withan immunogenically effective dose of RSV vaccine, typically in a volumeof 0.5 ml of a physiologically acceptable diluent or carrier. This hasthe advantage of simplicity and safety compared to parenteralimmunization with a non-replicating vaccine. It also provides directstimulation of local respiratory tract immunity, which plays a majorrole in resistance to RSV. Further, this mode of vaccination effectivelybypasses the immunosuppressive effects of RSV-specificmaternally-derived serum antibodies, which typically are found in thevery young. Also, while the parenteral administration of RSV antigenscan sometimes be associated with immunopathologic complications, thishas never been observed with a live virus.

[0209] In all subjects, the precise amount of gene position-shifted RSVvaccine administered and the timing and repetition of administrationwill be determined based on the patient's state of health and weight,the mode of administration, the nature of the formulation, etc. Dosageswill generally range from about 10³ to about 10⁶ plaque forming units(PFU) or more of virus per patient, more commonly from about 10⁴ to 10⁵PFU virus per patient. In any event, the vaccine formulations shouldprovide a quantity of attenuated RSV of the invention sufficient toeffectively stimulate or induce an anti-RSV immune response, e.g., ascan be determined by complement fixation, plaque neutralization, and/orenzyme-linked immunosorbent assay, among other methods. In this regard,individuals are also monitored for signs and symptoms of upperrespiratory illness. As with administration to chimpanzees, theattenuated virus of the vaccine grows in the nasopharynx of vaccinees atlevels approximately 10-fold or more lower than wild-type virus, orapproximately 10-fold or more lower when compared to levels ofincompletely attenuated RSV.

[0210] In neonates and infants, multiple administration may be requiredto elicit sufficient levels of immunity. Administration should beginwithin the first month of life, and at intervals throughout childhood,such as at two months, six months, one year and two years, as necessaryto maintain sufficient levels of protection against native (wild-type)RSV infection. Similarly, adults who are particularly susceptible torepeated or serious RSV infection, such as, for example, health careworkers, day care workers, family members of young children, theelderly, individuals with compromised cardiopulmonary function, mayrequire multiple immunizations to establish and/or maintain protectiveimmune responses. Levels of induced immunity can be monitored bymeasuring amounts of neutralizing secretory and serum antibodies, anddosages adjusted or vaccinations repeated as necessary to maintaindesired levels of protection. Further, different vaccine viruses may beindicated for administration to different recipient groups. For example,an engineered gene position-shifted RSV strain expressing a cytokine oran additional protein rich in T cell epitopes may be particularlyadvantageous for adults rather than for infants. RSV vaccines producedin accordance with the present invention can be combined with virusesexpressing antigens of another subgroup or strain of RSV to achieveprotection against multiple RSV subgroups or strains. Alternatively, thevaccine virus may incorporate protective epitopes of multiple RSVstrains or subgroups engineered into one RSV clone as described herein.

[0211] Typically when different vaccine viruses are used they will beadministered in an admixture simultaneously, but they may also beadministered separately. For example, as the F glycoproteins of the twoRSV subgroups differ by only about 10% in amino acid sequence, thissimilarity is the basis for a cross-protective immune response asobserved in animals immunized with RSV or F antigen and challenged witha heterologous strain. Thus, immunization with one strain may protectagainst different strains of the same or different subgroup. However,optimal protection probably will require immunization against bothsubgroups.

[0212] The gene position-shifted RSV vaccines of the invention elicitproduction of an immune response that is protective against seriouslower respiratory tract disease, such as pneumonia and bronchiolitiswhen the individual is subsequently infected with wild--type RSV. Whilethe naturally circulating virus is still capable of causing infection,particularly in the upper respiratory tract, there is a very greatlyreduced possibility of rhinitis as a result of the vaccination andpossible boosting of resistance by subsequent infection by wild-typevirus. Following vaccination, there are detectable levels of hostengendered serum and secretory antibodies which are capable ofneutralizing homologous (of the same subgroup) wild-type virus in vitroand in vivo. In many instances the host antibodies will also neutralizewild-type virus of a different, non-vaccine subgroup.

[0213] Preferred gene position-shifted RSV of the present inventionexhibit a very substantial diminution of virulence when compared towild-type virus that is circulating naturally in humans. The geneposition-shifted virus is sufficiently attenuated so that symptoms ofinfection will not occur in most immunized individuals. In someinstances the attenuated virus may still be capable of dissemination tounvaccinated individuals. However, its virulence is sufficientlyabrogated such that severe lower respiratory tract infections in thevaccinated or incidental host do not occur.

[0214] The level of attenuation of gene position-shifted RSV vaccinevirus may be determined by, for example, quantifying the amount of viruspresent in the respiratory tract of an immunized host and comparing theamount to that produced by wild-type RSV or other attenuated RSV whichhave been evaluated as candidate vaccine strains. For example, theattenuated chimeric virus of the invention will have a greater degree ofrestriction of replication in the upper respiratory tract of a highlysusceptible host, such as a chimpanzee, compared to the levels ofreplication of wild-type virus, e.g., 10- to 1000-fold less. Also, thelevel of replication of the attenuated RSV vaccine strain in the upperrespiratory tract of the chimpanzee should be less than that of the RSVA2 ts-1 mutant, which was demonstrated previously to be incompletelyattenuated in seronegative human infants. In order to further reduce thedevelopment of rhinorrhea, which is associated with the replication ofvirus in the upper respiratory tract, an ideal vaccine candidate virusshould exhibit a restricted level of replication in both the upper andlower respiratory tract. However, the attenuated viruses of theinvention must be sufficiently infectious and immunogenic in humans toconfer protection in vaccinated individuals. Methods for determininglevels of RS virus in the nasopharynx of an infected host are well knownin the literature. Specimens are obtained by aspiration or washing outof nasopharyngeal secretions and virus quantified in tissue culture orother by laboratory procedure. See, for example, (Belshe et al., J. Med.Virology 1:157-162, 1977; Friedewald et al., J. Amer. Med. Assoc.204:690-694, 1968; Gharpure et al., J. Virol. 3:414-421, 1969; andWright et al., Arch. Ges. Virusforsch. 41:238-247, 1973), eachincorporated herein by reference. The virus can conveniently be measuredin the nasopharynx of host animals, such as chimpanzees.

[0215] In some instances it may be desirable to combine the geneposition-shifted RSV vaccines of the invention with vaccines whichinduce protective responses to other agents, particularly otherchildhood viruses. For example, a gene position-shifted RSV vaccine ofthe present invention can be administered simultaneously withparainfluenza virus vaccine, such as described in Clements et al., J.Clin. Microbiol. 29:1175-1182, 1991), which is incorporated herein byreference. In another aspect of the invention the chimeric RSV can beemployed as a vector for protective antigens of other respiratory tractpathogens, such as PIV, by incorporating the sequences encoding thoseprotective antigens into the gene position-shifted RSV genome orantigenome which is used to produce infectious recombinant RSV, asdescribed herein.

[0216] In yet another aspect of the invention a gene position-shiftedRSV is employed as a vector for transient gene therapy of therespiratory tract. According to this embodiment the geneposition-shifted RSV genome or antigenome incorporates a sequence whichis capable of encoding a gene product of interest. The gene product ofinterest is under control of the same or a different promoter from thatwhich controls RSV expression. The infectious RSV produced bycoexpressing the recombinant RSV genome or antigenome with the N, P, Land M2(ORF1) proteins and containing a sequence encoding the geneproduct of interest is administered to a patient. This can involve arecombinant RSV which is fully infectious (i.e., competent to infectcultured cells and produce infectious progeny), or can be a recombinantRSV which, for example, lacks one or more of the G, F and SH surfaceglycoprotein genes and is propagated in cells which provide one or moreof these proteins in trans by stable or transient expression. In such acase, the recombinant virus produced would be competent for efficientinfection, but would be highly inefficient in producing infectiousparticles. The lack of expressed cell surface glycoproteins also wouldreduce the efficiency of the host immune system in eliminating theinfected cells. These features would increase the durability and safetyof expression of the foreign gene.

[0217] With regard to gene therapy, administration is typically byaerosol, nebulizer, or other topical application to the respiratorytract of the patient being treated. Gene position-shifted RSV isadministered in an amount sufficient to result in the expression oftherapeutic or prophylactic levels of the desired gene product. Examplesof representative gene products which are administered in this methodinclude those which encode, for example, those particularly suitable fortransient expression, e.g., interleukin-2, interleukin-4,gamma-interferon, GM-CSF, G-CSF, erythropoietin, and other cytokines,glucocerebrosidase, phenylalanine hydroxylase, cystic fibrosistransmembrane conductance regulator (CFTR), hypoxanthine-guaninephosphoribosyl transferase, cytotoxins, tumor suppressor genes,antisense RNAs, and vaccine antigens.

[0218] The following examples are provided by way of illustration, notlimitation.

EXAMPLE I

[0219] Construction of Recombinant RSV in which the G and F Genes HaveBeen Rearranged to a Promoter-Proximal Position Singly or in Combination

[0220] The present example documents rearrangement of the gene order ofinfectious RSV whereby one or more gene(s) or genome segment(s) encodingan antigenic determinant, exemplified by the G and/or F gene(s), isshifted to a more promoter-proximal position. In one example presentedbelow, the G and F genes are moved coordinately from their wild typegene order positions to occupy rearranged positions 1 and 2 of therecombinant RSV genome or antigenome. This manipulation was performedwith a cloned cDNA of RSV antigenomic RNA from which the SH gene wasdeleted (RSV ASH), as described above (see, e.g., Whitehead et al., J.Virol. 73:3438-3442, 1999, incorporated herein by reference). Wild typeRSV from which the SH gene is deleted grows as well or slightly betterthan complete wild type RSV in vitro, and is slightly attenuated in theupper respiratory tract of mice and in the upper and lower respiratorytracts of chimpanzee (Bukreyev et al., J. Virol. 71:8973-8982, 1997;Whitehead et al., J. Virol. 73:3438-3442, 1999, incorporated herein byreference). Its levels of immunogenicity and protective efficacy areclosely comparable to those of wild type virus. Thus, RSV ASH virus isslightly attenuated compared to wild type virus but otherwise has verysimilar biological properties, and it represents the parent virus inthese studies. These characteristics of the ASH virus, combined with theincreased genetic stability of RSV deletion mutants in general, renderthis background particularly useful as a parent recombinant forproduction of gene position-shifted RSV, facilitating recovery andmanipulation of mutant derivatives.

[0221] The antigenomic cDNA was manipulated and recombinant virusrecovered using procedures and strategies described above (see also,Collins et al., Proc. Natl. Acad. Sci. USA 92:11563-11567, 1995;Bukreyev et al., J. Virol. 70:6634-6641, 1996; Bukreyev et al., J.Virol. 71:8973-8982, 1997; Whitehead et al., J. Virol. 72:4467-4471,1998; Whitehead et al., Virology 247:232-239, 1998; Bermingham andCollins, Proc. Natl. Acad. Sci. USA 96:11259-11264, 1999; Collins etal., Virology 259:251-255, 1999; Bukreyev et al., Proc. Natl. Acad. Sci.USA 96:2367-2372, 1999; Juhasz et al., Vaccine 17:1416-1424, 1999;Juhasz et al., J. Virol. 73:5176-5180, 1999; Teng and Collins, J. Virol.73:466-473, 1999; Whitehead et al., J. Virol. 73:9773-9780, 1999;Whitehead et al., J. Virol. 73:871-877, 1999; Whitehead et al., J.Virol. 73:3438-3442, 1999; U.S. Pat. No. 5,993,824; each incorporatedherein by reference). The antigenomic cDNA was manipulated as threesubclones. One subclone, D51/ΔSH, contains the T7 promoter and left handend of the genome from the leader region to the downstream end of the Mgene. The second subclone, pUC19-GFM2, contains the G, F and M2 genesfrom the middle of the genome. The third subclone, D39, contains the Lgene followed by the trailer from the right hand end of the genomefollowed by a self-cleaving ribosome sequence and tandem T7transcription terminators.

[0222] PCR with mutagenic primers was used to amplify and modify theends of cDNAs containing the G and F genes separately or together as asingle G-F cDNA (FIG. 1). To make a cDNA of G alone for insertion, PCRwas used to amplify nucleotides 4692-5596 of the complete recombinantRSV antigenomic sequence (spanning from the ATG that initiates the G ORFto the downstream end of the G gene-end signal). The PCR primers weredesigned to add, immediately after the G gene-end signal, the first 6nucleotides of the G-F intergenic (IG) region followed by a copy of the10-nucleotide GS signal of the NS1 gene (FIG. 1). The PCR primers alsowere designed to add a BlpI site at both ends of the amplified cDNA. Tomake a cDNA of the F gene alone for insertion, PCR was used to amplifynucleotides 5662-7551 of the complete antigenomic sequence (spanningfrom the ATG that initiates the F ORF to the end of the F gene-endsignal). The PCR primers were designed to add, immediately after the Fgene-end signal, the first 6 nucleotides of the F-M2 IG followed by acopy of the 10-nucleotide NS1 gene-start signal. The PCR primers alsoplaced a BlpI site on both ends of the cDNA.

[0223] To make a cDNA containing the G and F genes for insertion, PCRwas used to amplify nucleotides 4692-7551 of the complete antigenomiccDNA (spanning from the ATG of the G ORF to the end of the F gene-endsignal) (FIG. 1). The PCR primers were designed to add, immediatelyafter the F gene-end signal, the first 6 nucleotides of the F-M2 IGfollowed by a copy of the NS1 gene-start signal. The PCR primers alsowere designed to add a BlpI site on both ends of the cDNA. All cDNAswere sequenced in their entirety to confirm structures.

[0224] In order to make a promoter-proximal site for insertion of the G,F, or G-F cDNA, the upstream noncoding region of the NS1 gene wasmodified by nucleotide substitutions at antigenome positions 92 (G to C,positive-sense) and 97 (A to C), thereby creating a BlpI site (FIG. 1).This manipulation was performed on a BstBI-MfeI subclone containing thefirst 419 nucleotides of the RSV antigenomic RNA. The nucleotidesubstitutions were introduced by PCR on complete plasmid (Byrappa etal., Genome Research 5:404-407, 1995; incorporated herein by reference).The modified BstBI-MfeI fragment was reinserted into D51/ΔSH, and thissubclone served in turn as the recipient for the G, F, and G-F BlpI cDNAfragments constructed as described above. Because Blpl has an asymmetricheptameric recognition sequence, the fragments can only be inserted inthe correct orientation.

[0225] When the G, F, or G-F cDNA was placed in the promoter-proximalposition, the corresponding gene(s) was/were deleted from the normaldownstream position, so that each recombinant genome incorporated asingle copy of G and F (FIG. 1). To delete G alone, PCR was performed onpUC19-G-F-M2 to amplify the StuI-HpaI fragment (nucleotides 5611-6419,spanning from the G-F IG into the middle of the F gene). In addition,this PCR added the sequence TTAATTAAAAACATATTATCACAAA (SEQ ID NO: 3) tothe upstream end of the CDNA. This sequence contains a PacI site(italicized), which in the antigenomic cDNA is located within the SHgene-end signal. This piece could then be introduced as a PacI-HpaIfragment into the PacI-HpaI window of unmodified pUC19-GFM2, therebydeleting the G gene. The sequence of the cDNA fragment that had beensubjected to PCR was confirmed by dideoxynucleotide sequencing.

[0226] Alternatively, to delete F alone, PCR was performed on pUC19-GFM2to amplify the fragment that runs from the PacI site at nucleotide 4618to the G gene-end signal at nucleotide 5596. In addition, this PCR addedthe sequence CACAATTGCATGC (SEQ ID NO: 4) to the downstream end of thecDNA. This sequence contained the upstream part of the F-M2 IG sequencefollowed by an SphI site (italicized) that is present in the F-M2 IG ofthe recombinant RSV antigenome. Cloning this cDNA as a PacI-SphIfragment into the PacI-SphI window of unmodified pUC19-GFM2 resulted indeletion of the F gene. The sequence of the cDNA fragment that had beensubjected to PCR was confirmed by dideoxynucleotide sequencing.

[0227] To delete both the G and F genes, the SphI-BamHI fragment, PCR bythe method of Byrappa (Byrappa et al., Genome Research, 5:404-407(1995)) et al. was used to amplify nucleotides 7559-8506 of pUC-GFM2(spanning from the F-M2 IG to the M2/L overlap) with the followingsequence added to the upstream end of the cDNA: TTAATTAAAAACACAATT (SEQID NO: 5). The resulting cDNA insert contains a PacI site (italicized)and the upstream part of the F-M2 IG sequence that immediately precedesthe SphI site, but lacks the G and F genes. The sequence of the cDNAfragment that had been subjected to PCR was confirmed bydideoxynucleotide sequencing.

[0228] Complete antigenomic cDNAs were then assembled as described above(see also, Collins et al., Proc. Natl. Acad. Sci. USA 92:11563-11567,1995; Bukreyev et al., J. Virol. 70:6634-6641, 1996; Bukreyev et al., J.Virol. 71:8973-8982, 1997; Whitehead et al., J. Virol. 72:4467-4471,1998; Whitehead et al., Virology 247:232-239, 1998; Bermingham andCollins, Proc. Natl. Acad. Sci. USA 96:11259-11264, 1999; Collins etal., Virology 259:251-255, 1999; Bukreyev et al., Proc. Natl. Acad. Sci.USA 96:2367-2372, 1999; Juhasz et al., Vaccine 17:1416-1424, 1999;Juhasz et al., J. Virol. 73:5176-5180, 1999; Teng and Collins, J. Virol.73:466-473, 1999; Whitehead et al., J. Virol. 73:9773-9780, 1999;Whitehead et al., J. Virol. 73:871-877, 1999; Whitehead et al., J.Virol. 73:3438-3442, 1999; and U.S. Pat. No. 5,993,8244; eachincorporated herein by reference). This assembly yielded cDNAs encodingBlp/ΔSH, G1/ΔSH, F1/ΔSH, and G1F2/ΔSH antigenomic cDNAs.

[0229] These cDNAs were transfected individually into HEp-2 cellstogether with N, P, M2-1 and L support plasmids and incubated at 32° C.(see, e.g., Collins et al., Proc. Natl. Acad. Sci. USA 92:11563-11567,1995; Collins et al., Virology 259:251-255, 1999; U.S. Pat. No.5,993,824, each incorporated herein by reference). The transfectionsupernatants were passaged to fresh cells 3 days later, and thensubjected to serial passage in HEp-2 cells at 37° C. with intervals ofharvest of 3 to 7 days. The shorter time intervals were necessary formonolayers infected with the F1/ΔSH and G1F2/ΔSH viruses because theyexhibited a more rapid development of syncytia and subsequent celldestruction. This was interpreted to reflect increased expression of thefusogenic F protein due to the alteration in F gene position. Aliquotsof each harvested supernatant were flash-frozen and titrated later inparallel, as shown in FIG. 2. These results indicate that the efficiencyof recovery and amplification of the G1F2/ΔSH and F1/ΔSH virusesexceeded those of the Blp/ΔSH or G1/ΔSH virus, as was also determinedrelative to the ΔSH virus and wild type virus.

[0230] Total RNA was isolated from cells infected with each of therecombinant viruses, and RT-PCR of appropriate genome segments wasperformed which confirmed that the engineered genomic structures were asdesigned and constructed. In addition, Northern blot analysis confirmedexpression of the appropriate subgenomic mRNAs.

[0231] The following viruses were then compared with regard to kineticsof growth and antigen production in vitro: wild type RSV (containing theSH gene), ΔSH (with the SH gene deleted but not containing a BlpI sitein the NS1 noncoding region), Blp/ΔSH, G1/ΔSH, and G1F2/ΔSH. Replicatemonolayer cultures of HEp-2 cells and Vero cells were infected at an MOIof 0.1 plaque forming units (PFU) per cell with an adsorption period of1 h (−1 to 0 hours). The monolayers were then washed three times andincubated at 37° C. Two duplicate monolayers per virus were harvested at12 h intervals, beginning immediately post-adsorption at t=0 hours. Themedium supernatants were flash-frozen for analysis later by plaque assayto quantitate released infectious virus (FIG. 3A). This showed that theviruses were comparable in their ability to produce infectious virus inVero (FIG. 3A, upper panel) and HEp-2 (FIG. 3A, lower panel) cells.

[0232] In the same experiment, the Vero cell monolayers from each timepoint (FIG. 3A, upper panel) were harvested and analyzed by Westernblotting to characterize the expression of G protein (FIG. 4). A sampleof total infected-cell protein from each time point was electrophoresedin denaturing polyacrylamide gel, transferred to nitrocellulose, andanalyzed by incubation with an antiserum specific to a peptide of the Gprotein. Bound antibodies were detected and quantitated using acommercial chemiluminescence kit. The antibody bound to the two majorcell-associated forms of the G protein, namely the larger 90 kDafully-mature form and the smaller 50 kDa incompletely-glycosylated form.This comparison showed that, although the production of infectiousparticles was similar for all viruses (FIG. 3A, upper panel), the amountof cell-associated G protein was considerably greater in cells infectedwith the G1/ΔSH or G1F2/ΔSH virus (FIG. 4). Quantitation of the gelbands by densitometry of the exposed filn indicated that the G1/ΔSH andG1F2/ΔSH viruses expressed 6-fold and 4-fold, respectively, more Gprotein than did the Blp/ΔSH virus.

[0233] Cell monolayers infected with the F1/ΔSH or G1F2/ΔSH virusesexhibited a rapid onset of cytopathic effect involving syncytiumformation, as mentioned above. This was interpreted to reflect increasedexpression of the F protein. This enhanced cytopathic effect would notcontraindicate the use of this virus as a vaccine, since infection invivo with a vaccine virus involves only a small amount of epithelialcells which die and are replaced whether the onset of cytopathogenicityfor these scattered cells is more rapid or not. However, it may be thathigher titers of virus might have been achieved if the cytopathogenicityin vitro had been less rapid. Resolution of these factors will beachieved by construction of gene position-shifted RSV in backgroundsthat are more highly attenuated. It is notable in this context that thegene shifts did not interfere with the growth of RSV but did increasethe total expression of G protein by several fold.

[0234] In further studies, the ability of the gene-shift viruses toreplicate in HEp-2 and Vero cell monolayers was compared in anexperiment in which the multiplicity of infection was 3.0 (FIG. 3B).This was done in two separate experiments that yielded similar results.Data for one of the experiments are shown in FIG. 3B. In thissingle-cycle growth assay, each of the three viruses containing G and/orF in the promoter-proximal position, namely G1/ΔSH, F1/ΔSH, andG1F2/ΔSH, replicated more efficiently than the control virus Blp/SH.Furthermore, the difference was greater in Vero cells than in HEp-2cells. For example, at 24 h post infection, the titer of the G1/ΔSHvirus was 7.2×10⁶ PFU/ml, compared to 6.8×10⁶ PFU/ml for the Blp/ΔSHcontrol in HEp-2 cells, a difference of 0.4 log₁₀ whereas the respectivevalues in Vero cells were 6.6×10⁶ PFU/ml for the G1/ΔSH virus and5.6×10⁶ PFU/ml for the Blp/ΔSH control virus, a difference of 1.0 log₁₀.Each of the other gene-shift viruses also had titers in excess of thatof the control virus. Thus, the shift of G and/or F to the promoterproximal position provided, in this example, an increase in virus yieldin both HEp-2 and Vero cells, with a maximum yield of 10-fold in Verocells, which is a cell line that is useful for large-scale virusproduction.

[0235] The gene-shift recombinant viruses also were examined for theability to replicate in the upper and lower respiratory tract of BALB/cmice (Table 1). Mice in groups of 18 animals each were infected with 106PFU per animal of G1/ΔSH, F1/ΔSH, G1F2/ΔSH, or the control virusBlp/ΔSH. Six animals from each group were sacrificed on days 3, 4 and 5post-infection, and the nasal turbinates and lungs were harvested andanalyzed by plaque assay to determine virus titers in the upper andlower respiratory tracts, respectively. As shown in Table 1, the levelof replication of the Blp/ΔSH and F1/ΔSH viruses were essentiallyindistinguishable. Thus, although the latter virus replicated somewhatmore efficiently in vitro, its replication in vivo was not changed. Theabsence of increased replication or virulence would simplifymodification of the gene-shift viruses by the addition of attenuatingmutations. The replication of the G1F2/ΔSH virus was marginally lowerthan that of the “wild type” Blp/ΔSH control, and the replication of theG1/ΔSH virus also was lower, particularly early in the infection. Forexample, on day 3 the G1/ΔSH virus was 0.7 log₁₀ lower in both the upperand lower respiratory tract compared to the Blp/ΔSH control. TABLE 1Replication, in the upper and lower respiratory tract of mice, ofrecombinant RSV containing the G and/or F genes in the promotor-proximalposition. Nasal Turbinates Lungs Mean mean titer ± SE titer ± SE (log₁₀PFU/g tissue) (log₁₀ PFU/g tissue) Virus¹ Day 3 Day 4 Day 5 Day 3 Day 4Day 5 B1p/ΔSH 4.2 ± 0.17 4.0 ± 0.32 3.5 ± 0.2  3.6 ± 0.26 4.1 ± 0.37 4.5± 0.09 G1/ΔSH 3.5 ± 0.24 3.4 ± 0.54 3.5 ± 0.24 2.9 ± 0.2  3.9 ± 0.34 4.6± 0.13 F1/ΔSH 4.4 ± 0.11 4.1 ± 0.1  3.9 ± 0.12 3.9 ± 0.09 4.7 ± 0.08 4.9± 0.16 G1F2/ΔSH 3.3 ± 0.50 3.5 ± 0.13 3.3 ± 0.13 3.1 ± 0.13 4.2 ± 0.284.1 ± 0.07

[0236] 1. BALB/C mice in groups of 18 were inoculated intranasally with10⁶ PFu per mouse of the indicated virus on day 0. On days 3, 4, and 5,six mice per group were sacrificed and the nasal turbinates and lungswere harvested and virus titers were determined by plaque assay. Meantiters are shown with the standard error indicated.

[0237] Thus, shifting one or more genes to the promoter proximalposition, or rearranging RSV genes in general, can modestly attenuatethe virus for replication in vivo. This has value for designing anattenuated vaccine strain, since it adds to the menu of useful methodsof attenuation. Furthermore, it is important to have attenuatingmutations that represent different classes or types, such astemperature-sensitive point mutations, non-temperature-sensitive pointmutations, gene deletions, and so forth. Different types of mutationoperate in different ways to affect viral phenotype, and the presence ofmultiple types of mutations in a single vaccine virus confers increasedstability. Attenuation by gene shift represents an additional usefulclass of attenuating mutations in this context. The finding that geneshifts according to the invention can confer a modest degree ofattenuation provides useful new tools for fine-tuning the attenuationphenotype of vaccine strains.

[0238] Certain useful attenuating mutations within the invention are ofa “conditional” variety, where attenuation is minimal under specifiedconditions (e.g., in vitro) and maximal under other conditions (e.g., invivo). An attenuation phenotype that is minimal or not operant in vitropermits efficient production of vaccine virus, which is particularlyimportant in the case of RSV and other viruses that grow poorly in cellculture. Of course, as illustrated here, the attenuation phenotype mustbe operant in vivo in order to reduce disease and reactogenicity of thevaccine virus.

[0239] The G1/ΔSH recombinant shown here illustrates a particularlydesirable combination of traits resulting from the gene-shift.Specifically, its growth in vitro actually was increased up to 10-fold,while its replication in vivo was decreased moderately. This providesthe advantage of improved efficiency of vaccine production inconjunction with attenuation in vivo, and improved antigen expression,as described above.

[0240] The immunogenicity of the gene-shift viruses in vivo wasinvestigated in BALB/c mice. Mice in groups of six were infected withthe individual viruses as described immediately above, and serum sampleswere taken 1 day prior to inoculation and 28 and 56 days postinoculation (Table 2). The serum samples were analyzed byglycoprotein-specific enzyme-linked immunoadsorbent assay (ELISA)specific to IgG (Table 2). Analysis of G protein-specific IgG showedthat the responses to the F1/ΔSH and G1F2/ΔSH viruses were very similarto that for the Blp/ΔSH control virus. On the other hand, the G-specificresponse to the G1/ΔSH virus moderately decreased (up to four-fold).This likely is due at least in part to the reduced replication of thisvirus, as described above in Table 1. Analysis of F-specific responsesshowed that the F1/ΔSH and G1F2/ΔSH viruses had moderate increases (2.5-to 4-fold) in antibody levels compared to the G1/ΔSH and Blp/ΔSHviruses, which is consistent with the interpretation that moving the Fgene to the promoter-proximal position results in increased antigenexpression in vivo and increased immunogenicity. Furthermore, theseresults indicated that the gene-shift can result in increasedimmunogenicity in vivo under conditions where overall replication of theimmunizing virus is not changed. This is a highly desirable outcome,since it provides a specific method to make an RSV vaccine that is Δisinherently more immunogenic than the wild type parent virus. It shouldbe noted that the mouse model can be used to identify and characterizebiological properties of a virus and can reveal desirable new featuressuch as shown here. TABLE 2 Measurement, by glycoprotein-specificenzyme-linked immunoabsorbent assay (ELISA), or serum antibody responsesin mice following infection with recombinant RSV containing G and/or Fgene in the promotor-proximal position. Serum immunoglobulin G ELISAtiter (mean reciprocal log₂ ± SE) against indicated RSV protein² No. ofanimals Anti RSV Anti RSV per G IgG F IgG Virus¹ group Pre Day 28 Day 56Pre Day 28 Day 56 B1p/ΔSH 6 ≦5.3 ± 0 9.6 ± 0.6 11.7 ± 0.8 ≦5.3 ± 0 10.3± 0.4 12.0 ± 0.4 G1ΔSH 6 ≦5.3 ± 0 9.0 ± 0.6  9.6 ± 0.6 ≦5.3 ± 0 10.0 ±0.4 12.0 ± 0.7 F1ΔSH 6 ≦5.3 ± 0 9.6 ± 0.9 11.6 ± 0.6 ≦5.3 ± 0 11.6 ± 0.313.6 ± 0.3 G1F2ΔSH 6 ≦5.3 ± 0 9.3 ± 0.7 11.6 ± 0.8 ≦5.3 ± 0 12.3 ± 0.414.0 ± 0.4

[0241] 1. BALB/C mice in groups of six were inoculated intranasally with10⁶ PFU of virus in a 0.1 ml inoculum on day 0.

[0242]2. Serum samples were taken 1 day prior to inoculation (Pre) and28 and 56 days post inoculation, and were analyzed byglycoprotein-specific ELISA for IgG antibodies against the RSV G or Fprotein, as indicated. Mean titers are shown with standard errorindicated.

EXAMPLE II

[0243] Recombinant RSV Incorporating the G and F genes in aPromoter-Proximal Shifted Position in a Highly Attenuated Background

[0244] The gene shift mutations described above were introduced in thecontext of a genetic background from which the SH gene was deleted. Inwild type virus, this deletion modestly improves growth in cell culture,and is moderately attenuating in vivo (Bukreyev et al., J. Virol.71:8973-8982, 1997; Whitehead et al., J. Virol. 73:3438-3442, 1999,incorporated herein by reference). However, an RSV vaccine virus that issafe for administration to RSV-naive infants and children requiresfurther attenuation than is provided by the ΔSH deletion alone.Therefore, the present example is provided to demonstrate that geneshift mutants of the invention can be recovered successfully in a highlyattenuated background.

[0245] Two backgrounds were chosen to exemplify this aspect of theinvention, one lacking both the SH and NS2 genes and one lacking the SH,NS1 and NS2 genes. As described in the above-incorporated references,the ΔNS2 and ΔNS 1 deletions are each highly attenuating on its own(see, e.g., Whitehead et al., J. Virol. 73:3438-3442, 1999). In vitro,the production of virus containing either mutation is delayed andreduced, although under vaccine production conditions a yield of ΔNS2virus is achieved comparable to that of wild type virus. In chimpanzees,the ΔNS2 and ΔNS1 viruses each are highly attenuated for replication anddisease and are highly immunogenic and protective against RSV challenge.Each mutation alone is an excellent candidate to be included in arecombinant vaccine virus, either on its own or in combination withother mutations. The ΔNS1 and ΔNS2 mutations together result in a virusthat is even more highly attenuated in vitro than the ΔNS2 virus. In thepresent example, further combinations of these deletions wereconstructed and tested for their ability to support further geneposition-shifted mutations.

[0246] Antigenomic cDNAs were constructed in which the G and F geneswere moved to positions 1 and 2 of an antigenome from which the NS2 andSH genes had been deleted, designated G1F2/ΔNS2ΔSH (FIG. 5, panel A), orto positions 1 and 2 of an antigenome in which the NS1, NS2 and SH geneswere deleted, designated G1F2/ΔNS1ΔNS2ΔSH (FIG. 5, panel B). Theseantigenomic cDNAs were used to recover recombinant virus as described inExample I above. In both cases, recombinant virus was readily recoveredand propagated in vitro. Thus, the present example demonstrates thatgene position-shifted RSVs containing multiple attenuating mutationswith the G and F genes shifted to the promoter-proximal positions can bereadily produced and recovered. These and other gene position-shiftedRSVs will be analyzed for levels of replication and antigen expression,as well as growth, immunogenicity and protective efficacy in vivo toselect suitable vaccine candidates in accordance with the methodsdescribed herein.

[0247] In the foregoing examples, representative changes were made inthe gene order of RSV to improve its properties as a live-attenuatedvaccine. In particular, the G and F genes were moved, singly and intandem, to a more promoter-proximal position. These two proteinsnormally occupy positions 7 (G) and 8 (F) in the RSV gene order(NS1-NS2-N-P-M-SH-G-F-M2-L). In order to increase the possibility ofsuccessful recovery, the manipulations were performed in a version ofRSV in which the SH gene had been deleted. G and F were then movedindividually to position 1, or were moved together to positions 1 and 2,respectively. Surprisingly, recombinant RSV were readily recovered inwhich G or F were moved to position 1, or in which G and F were moved topositions 1 and 2, respectively. This result differed greatly fromprevious studies with VSV, where movement of the single VSV glycoproteingene by only two positions was very deleterious to virus growth. Theability to recover these altered viruses also was surprising because RSVreplicates inefficiently and because RSV has a complex gene order andmovement of the glycoprotein genes involved a large number of positionchanges. Indeed, the rearranged RSV's grow at least as well as doestheir immediate parent having the wild type order of genes. As indicatedabove, this is particularly important for RSV, since the wild type virusgrows inefficiently in cell culture and a further reduction inreplication in vitro would likely render vaccine preparation unfeasible.It is remarkable that all of the NS1-NS2-N-P-M proteins could bedisplaced by one or two positions relative to the promoter without asignificant decrease in growth fitness. In addition, examination of theexpression of the G glycoprotein showed that it was increased up toseveral-fold over that of its parent virus. This indicated that avaccine virus containing G and/or F in the first position expresses ahigher molar amount of these protective antigens compared to the otherviral proteins, and thus represent a virus with highly desirable vaccineproperties.

[0248] Furthermore, the modification in gene order also was achievedwith two highly attenuated vaccine candidates produced in previous work,in which the NS2 gene was deleted on its own as described previously, orin which the NS1 and NS2 genes were deleted together. In these twovaccine candidates, the G and F glycoproteins were moved together topositions 1 and 2 respectively, and the G, F and SH glycoproteins weredeleted from their original downstream position. Thus, the recoveredviruses G1F2ΔNS2ΔSH and G1F2/ΔNS1ΔNS2ΔSH had two and three genes deletedrespectively in addition to the shift of the G and F genes. Toillustrate the extent of the changes involved, the gene orders of wildtype RSV (NS1-NS2-N-P-M-SH-G-F-M2-L) and the G1F2/ΔNS2ΔSH virus(G-F-NS1-N-P-M-M2-L) or the ΔNS1ΔNS2ΔSH (G-F-N-P-M-M2-L) can becompared. This shows that the positions of most or all of the genesrelative to the promoter were changed. Nonetheless, these highlyattenuated derivatives retained the capacity to be grown in cellculture, indicating their clear utility for development of candidatevaccine viruses.

EXAMPLE III

[0249] Construction of a Chimeric BRSV/HRSV Containing the HRSV G and FGenes in a Promoter-Proximal Shifted Position

[0250] The present example describes construction of an infectiousrBRSV/HRSV chimera in which the HRSV G and F genes are substituted intoa recombinant bovine RSV (rBRSV) background. The resulting human-bovinechimera contains two genes of HRSV, namely G and F, and eight genes fromBRSV, namely NS1, NS2, N, P, M, SH, M2 and L. Additional detaildescribing a human-bovine RSV construct having the human G and F genessubstituted at their corresponding, wild type positions in a bovine RSVbackground (designated rBRSV/A2) is provided in U.S. patent applicationSer. No. 09/602,212, filed by Bucholz et al. on Jun. 23, 2000, itscorresponding PCT application published as WO 01/04335 on Jan. 18, 2001,and its priority provisional U.S. application Ser. No. 60/143,132 filedon Jul. 9, 1999, each incorporated herein by reference.

[0251] In addition to the basic substituted glycoprotein construction ofrBRSV/A2, the HRSV G and F genes were shifted in the present example toa more promoter-proximal position in the rBRSV backbone relative to awild type gene order position of the F and G genes in the BRSV genome.More specifically, the F and G genes were moved from their usuallocation relative to the promoter, namely gene positions 7 and 8,respectively, to positions 1 and 2, respectively. To achieve thisobjective, complete infectious rBRSV was constructed in which nucleotidesubstitutions were made to create unique NotI, SalI and XhoI sites atpositions 67, 4,673 and 7,471, respectively (FIG. 6, panel A) (see also,Buchholz et al., J. Virol. 73:251-259, 1999; Buchholz et al., J. Virol.74:1187-1199, 2000, incorporated herein by reference). The NotI site iscontained within the upstream nontranslated region of the BRSV NS1 gene,and the SalI and XhoI sites are in intergenic regions. Digestion of therBRSV antigenomic cDNA with SalI and XhoI excised the BRSV G and F genesin their entirety and created compatible cohesive ends that were ligated(FIG. 6, panel B). This resulted in an rBRSV antigenomic cDNA lackingthe G and F genes and containing a 64-nucleotide SH-M2 intergenic regionwith the following sequence:

[0252] TTAAACTTAAAAATGGTTTATGtcgaGGAATAAAATCGATTAACAACCAATCAT TCAAAAAGAT(SEQ ID NO: 6) (the tetranucleotide cohesive ends of the originalcleaved SalI and XhoI sites are in small case). For comparison, thenaturally-occurring BRSV F-M2 intergenic sequence is 55 nucleotides inlength.

[0253] A cDNA containing the HRSV G and F genes was prepared by PCR withmutagenic primers used to modify the cDNA ends. Specifically, PCR wasused to amplify nucleotides 4692-7551 of the complete HRSV antigenomiccDNA (spanning from the ATG of the G ORF to the end of the F gene-endsignal), and the primers were designed to add, immediately after the Fgene-end signal, the first 6 nucleotides of the F-M2 IG followed by acopy of the NS1 gene-start signal. The PCR primers also were designed toadd a BlpI site and an NotI site each on both ends of the cDNA. Thesequence of the cDNA fragment that was subjected to PCR was confirmed bydideoxynucleotide sequencing. This cDNA was then inserted as a NotIfragment into the unique NotI site of the rBRSV antigenomic cDNA lackingthe G and F genes as described above. A correct recombinant wasidentified by restriction fragment mapping, and was designatedrBRSV/A2-G1F2. The structure of the encoded genomic RNA is shown in FIG.6, panel C. As shown, in this cDNA the G and F genes were moved frompositions 7 and 8 relative to the promoter to positions 1 and 2.

[0254] A plasmid encoding the antigenomic RNA of rBRSV/A2-G1 F2 wastransfected, together with plasmids encoding the N, P, M2-1 and Lsupport proteins, into BSR T7/5 cells, which stably express the T7 RNApolymerase, as described above (see also, Buchholz et al., J. Virol.73:251-259, 1999; Buchholz et al., J. Virol. 74:1187-1199, 2000, eachincorporated herein by reference), and infectious virus was recovered.The recovered rBRSV/A2-G1F2 virus was compared to rBRSV, rHRSV (alsocalled rA2) and rBRSV/A2 with regard to the efficiency of multicyclegrowth in human HEp-2 cells and bovine MDBK cells. As described above(see also, Buchholz et al, J.Virol. 74:1187-1199, 2000, incorporatedherein by reference), rHRSV grows much more efficiently than rBRSV inHEp-2 cells, and the rBRSV/A2 virus grows with an efficiencyintermediate between that of each parent. As shown in FIG. 7, theefficiency of replication of rBRSV/A2-G1F2 was indistinguishable fromthat of rBRSV/A2. Thus, unexpectedly, the change in the location of theG and F genes did not reduce the efficiency of growth in vitro, whichnovel result allows for efficient production of RSV vaccine virus.

[0255] Immunofluorescence was performed on HEp-2 cells infected with wtHRSV or the chimeric viruses rBRSV/A2 or rBRSV/A2-G1F2. This wasperformed using two monoclonal antibodies specific to the HRSV G or Fproteins, namely 021/01G and 44F, respectively (Lopez et al., J. Virol.72:6922-6928, 1998; Melero et al., J. Gen. Virol. 78:2411-2418, 1997,each incorporated herein by reference). The staining was done with eachmonoclonal antibody individually. Although this assay is onlysemi-quantitative, it has been previously determined that the assaydistinguishes reliably between wt rBRSV and rBRSV/A2 (the latter bearingthe HRSV G and F genes in the normal genome location in the rBRSVbackbone). In particular, wt HRSV gives a very strong, extensive patternof immunofluorescence indicative of efficient and extensive antigenexpression, while rBRSV/A2 gives a weaker, more diffuse, less extensivepattern (Buchholz et al., J. Virol. 74:1187-1199, 2000, incorporatedherein by reference). A comparable assay conducted for rBRSV/A2-G1F2(FIG. 8), shows that the pattern of immunofluorescence for thispromoter-shifted chimeric virus was very similar to that of wt HRSV.This result is consistent with increased expression of the G and Fglycoproteins. At the same time, the cytopathic effect associated withrBRSV/A2-G1F2 was reduced compared to wt HRSV, and more closelyresembled that of rBRSV/A2 (Buchholz et al., J. Virol. 74:1187-1199,2000, incorporated herein by reference). Specifically, rBRSV/A2 andrBRSV/A2-G1F2 induced fewer and smaller syncytia.

[0256] Thus, the present example documents modification of the rBRSV/A2human-bovine chimeric RSV virus, which contains the genes for the majorprotective antigens of HRSV, the G and F proteins, in the background ofBRSV which is strongly attenuated for replication in the respiratorytract of primates. The rBRSV/A2 virus has a strong host rangerestriction that renders it highly attenuated in primates. Since thepresent gene position-shifted rBRSV/A2-G1F2 virus bears the sameconstellation of BRSV genes in its genetic background, it is likely toshare this strong host range restriction phenotype, thereby increasingthe expression of the two major protective antigens. The increasedexpression of these two protective antigens in vivo is further expectedto increase the immunogenicity of this virus. Thus, the present examplemodified and improved the rBRSV/A2 virus by moving the HRSV genes to apromoter proximal location. A positional shift of this magnitude, i.e.,where the G and F genes were moved from wild type positions 7 and 8relative to the promoter to new positions 1 and 2, has not beendescribed previously.

EXAMPLE IV

[0257] Construction of a Chimeric BRSV/HRSV With Envelope-Associated M,G and F Proteins Derived From HRSV

[0258] The present example demonstrates yet another geneposition-shifted RSV generated within a human-bovine chimeric backgroundwhich involves modification of an antigenic chimeric virus resemblingthe rBRSV/A2 chimera (having the HRSV G and F protective antigen genesin a BRSV host-range-attenuated background), described above. Both BRSVand HRSV have 4 envelope-associated proteins: the G and F glycoproteinswhich are the major protective antigens; the small hydrophobic SHprotein of unknown function which does not appear to be a neutralizationor protective antigen for HRSV (Whitehead et al., J. Virol.73:3438-3442, 1999; Connors et al., J. Virol. 65:1634-1637, 1991, eachincorporated herein by reference); and the nonglycosylated internalmatrix M protein, which is not a protective antigen but is important invirion assembly (Teng and Collins, J. Virol. 72:5707-16, 1998,incorporated herein by reference).

[0259] In this example, a BRSV/HRSV chimeric virus was constructed inwhich all four BRSV envelope-associated protein genes were deleted,namely BRSV M, SH, G and F, and in which three HRSV envelope-associatedprotein genes, namely M, G and F, were inserted in their place. Thisyields a promoter-proximal gene shift of the F and G glycoprotein genesby the distance of one gene, corresponding to the length of the SH gene.

[0260] The above-described rBRSV/A2 construct (see also, Buchholz etal., J. Virol. 74:1187-1199, 2000, incorporated herein by reference) wasmodified to contain a unique MluI site at position 3204, within theintergenic region between the P and M genes (FIG. 9, panel A; P-M IG).This involved the introduction of 5 nucleotide substitutions. Nucleotidesequence position numbers are relative to the complete rBRSV antigenome(Buchholz et al., J. Virol. 73:251-259, 1999; Buchholz et al., J. Virol.74:1187-1199, 2000; GenBank accession number AF092942 or complete rHRSVantigenome in Collins et al., Proc. Natl. Acad. Sci. USA 92:11563-11567,1995; each incorporated herein by reference), and sequence positionnumbers that refer to the HRSV sequence are underlined. The MluI-SalIfragment was excised and replaced by the MluI-SalI fragment bearing theM gene.

[0261] Referring to FIG. 9, panel B, a cDNA containing the HRSV M genewas amplified and modified by PCR using primers that introduced changesto the ends of the cDNAs. Specifically, the M cDNA was modified so thatits upstream end contained an MluI site, followed by the last nucleotideof the P-M intergenic region (which is the same in HRSV and BRSV),followed by the complete HRSV M gene, followed by the first 4nucleotides of the BRSV SH-G intergenic region, followed by a SalI site.The sequence of this cDNA was confirmed to be correct in its entirety.It was digested with MluI and SalI and cloned into the MluI-SalI windowof the rBRSV antigenome. This resulted in rBRSV/A2-MGF. As shown in FIG.9, panel C, this chimera contained a backbone of six BRSV genes, namelyNS1, N52, N, P, M2 and L, and three HRSV envelope-associated proteingenes, namely M, G and F.

[0262] This antigenomic plasmid was transfected, together with plasmidsencoding the N, P, M2-1 and L support proteins, into BSR T7/5 cells,which stably express the T7 RNA polymerase, as described in detailpreviously (Buchholz et al., J. Virol. 73:251-259, 1999; Buchholz etal., J. Virol. 74:1187-1199, 2000, each incorporated herein byreference), and infectious virus was recovered. Thus, the presentexample also demonstrates that gene position-shifted RSVs containing agene deletion resulting in a promoter proximal shift of the G and Fgenes can be readily produced and recovered. This and other geneposition-shifted RSVs will be analyzed for levels of replication andantigen expression, as well as growth, immunogenicity and protectiveefficacy in vivo to select suitable vaccine candidates in accordancewith the methods described herein.

EXAMPLE V

[0263] Construction and Recovery of Additional BRSV/HRSV ChimericViruses Containing Nonstructural and/or Envelope-Associated Proteins ofHRSV Substituted Into the BRSV Backbone

[0264] Additional BRSV/HRSV chimeric viruses were constructed thatcontained HRSV nonstructural NS1 and NS2 genes and/orenvelope-associated M, SH, G and/or F genes substituted into the BRSVbackbone. Most of these chimeric viruses contained G and F genes derivedfrom HRSV, a desirable feature since these encode the major protectiveantigens and would be important for an effective HRSV vaccine.

[0265] In certain exemplary viruses, the BRSV NS1 and NS2 genes werereplaced by their HRSV counterparts. NS1 and NS2 have recently beenshown to be antagonists of the type I interferon-mediate antiviral state(Schlender, et al., J. Virol., 74:8234-42, 2000, incorporated herein byreference) and substitution of these genes offers a way of modifying thegrowth properties and virulence of a vaccine virus. This is due to thegeneral finding that interferon antagonists tend to be host specific(Young, et al., Virology, 269:383-90, 2000; Didcock, et al., J. Virol.,73:3125-33, 1999; Didcock, et al., J. Virol., 73:9928-33, 1999, eachincorporated herein by reference). Thus, inclusion of BRSV-specific NS1and NS2 genes in a vaccine virus would improve its growth in bovinecells but would constitute an attenuating mutation with regard to growthin primate cells and in the human vaccinee.

[0266] Conversely, HRSV-specific NS1 and NS2 genes would offer a way ofimproving the growth of a vaccine virus in human cells. Thus, thisprovides a new method for manipulating the growth properties andreactogenicity of a vaccine virus. In another virus, the completeconstellation of HRSV-specific membrane associated genes, namely M, SH,G and F, was placed in the BRSV backbone. Since the various proteins ofthe virus particle are thought to interact in various ways during geneexpression, genome replication, and virion production, the ability tomake a variety of combinations provides a rich source of vaccinecandidates.

[0267] Finally, this additional exemplary panel of viruses containedexamples of genes that were substituted without a change in gene order,others in which the gene order of the substituted viruses was altered,as well as ones in which some of the substituted genes did not have achange in order with regard to the BRSV backbone whereas others did.Thus, this panel provided a stringent test of the ability to manipulatecDNA-derived virus to make a wide array of chimeric viruses, and torecover viable viruses with altered and desirable biological properties.

[0268] A BRSV/HRSV chimeric virus was constructed in which the NS1 andNS2 genes of the rBRSV backbone were removed and replaced with the NS1and NS2 genes of HRSV, creating a virus called rBRSV/A2-NS1+2 (FIG. 10,second construct from the top). The backbone for this construction wasthe rBRSV antigenomic cDNA which had been modified to contain the uniqueNotl, Kpnl, Sall and Xhol sites illustrated in FIG. 10 (top construct)and described in detail previously (Buchholz, et al., J. Virol.,73:251-9, 1999; Buchholz, et al., J. Virol., 74:1187-1199, 2000, eachincorporated herein by reference). The HRSV NS1 and NS2 coding sequenceswere amplified by PCR as a single fragment which spanned positions 75 to1036 of the complete HRSV antigenomic sequence and included the HRSV NS1ORF, the NS1/N2 gene junction, and the NS2 ORF. The upstream end of thefragment also contained an added Notl site immediately before the HRSVsequence and a Blpl site added in the nontranslated sequence upstream ofthe NS1 ORF at HRSV position 91. The downstream end of the PCR cDNAcontained a Kpnl site immediately following the HRSV sequence. This PCRproduct was cloned and its sequence confirmed. It was then inserted as aNotl-Kpnl fragment into the corresponding window of the rBRSV backbone.

[0269] Another BRSV/HRSV chimeric virus was made in which the followingfour genes in rBRSV were replaced with their HRSV counterparts: NS1,NS2, G and F. This virus is designated rBRSV/A2-NS1+2GF (FIG. 10, thirdconstruct from the top). This construct was made by combining fragmentsfrom rBRSV/A2-NS1+2 and the previously described rBRSV/A2 (see, FIGS. 7and 8; U.S. patent application Ser. No. 09/602,212; Buchholz, et al., J.Virol. 74:1187-1199, 2000, each incorporated herein by reference), whichis optionally termed herein rBRSV/A2-GF. Specifically both constructscontained a Xmal site in the plasmid sequence upstream of the leaderregion and the Kpnl site illustrated in FIG. 10. The XmaI-Kpnl fragmentof rBRSV/A2-NS1+2 was transferred into the corresponding window of therBRSV/A2-GF plasmid.

[0270] Another BRSV/HRSV chimeric virus was made in which the followingfour genes in rBRSV were replaced by their HRSV counterparts: M, SH, Gand F. This virus was designated rBRSV/A2-MSHGF (FIG. 10, fourthconstruct from the top). This involved a rBRSV backbone in which a MluIsite was added in addition to the P-M intergenic region (see FIG. 9).Thus, the inserted HRSV sequence had as its upstream and downstreamboundaries the Mlul and Xhol sites shown in FIG. 9. This HRSV insert,bearing the M-SH-G-F sequence of HRSV flanked by Mlul and Xhol sites,was prepared by PCR, and the resulting product was cloned and itssequence confirmed. The Mlul-Xhol fragment was then cloned into thecorresponding window in the rBRSV backbone.

[0271] Another BRSV/HRSV chimeric virus was made in which the G and Fgenes were replaced by their HRSV counterpart placed in the third andfourth positions in the rBRSV backbone. This virus was designatedrBRSV/A2-G3F4 (FIG. 10, fifth construct from the top). For thisconstruction, PCR was used to amplify the HRSV G and F ORFs. The PCRprimers were designed to add to the upstream end of G the followingfeatures: a Kpn 1 site, a BRSV gene end signal (5′-AGTTATTTAAAAA) and athree nt intergenic sequence (CAT), which was then followed by the HRSVG and F genes. The downstream end of the amplified fragment ended in thedownstream nontranslated region of the HRSV F gene, at HRSV antigenomicposition 7420, followed by an added Kpn I site. This PCR product wascloned and its sequence confirmed. The Kpn I fragment bearing the HRSV Gand F sequence was then cloned into the Kpn I site of the rBRSV cDNAlacking the G and F genes as shown in FIG. 6, panel B. A recombinantcontaining the insert in the correct orientation was identified byrestriction analysis.

[0272] Another BRSV/HRSV chimeric virus was made in which the followinggenes were replaced in the rBRSV backbone: NS1, NS2, G and F, with the Gand F genes in the promoter-proximal position. This construct isdesignated HEx, or rBRSV/A2-G1F2NS3NS4 (FIG. 10, bottom construct). Thischimera was generated by modifying rBRSV/A2-NS1+2 to replace the BRSV Gand F genes by their HRSV counterparts in the first and second position,in the same way as described above for the construction of rBRSV/A2-G1F2(Example III, FIG. 6, panel B). Specifically, the BRSV G and F geneswere excised from the rBRSV/A2-NS1+2 antigenomic cDNA by digestion withSalI and XhoI, as described above (Example III). Subsequently, a NotIfragment containing the HRSV G and F genes as described above (ExampleIII, FIG. 6, panel B) was cloned into the singular NotI site which islocated immediately before the HRSV sequence in the NS1 noncoding regionof the rBRSV/A2-NS1+2 antigenomic cDNA. A recombinant containing theinsert in the correct orientation was identified by restrictionanalysis.

[0273] Each of the viruses listed above was readily recovered from cDNA,and in no case to date was a virus designed that could not be recovered.

[0274] The new rBRSV/A2-G3F4 and HEx viruses were compared for growthefficiency in vitro in parallel with the previously-tested rBRSV/A2-GFand rBRSV/A2-GF viruses (as noted above, the last virus was calledrBRSV/A2 in previous examples but was renamed here for clarity comparedto the new constructions). Monolayer cultures of Vero cells wereinfected at a multiplicity of infection of 0.1 and incubated at 37° C.Aliquots were taken at the time points shown in FIG. 11, and the virustiter was determined by plaque assay. Under these conditions, BRSVreplicates somewhat less efficiently than HRSV, reflecting its hostrange restriction in primate cells. As shown in FIG. 11, top panel, therBRSV/A2-G3F4 exhibited improved growth in vitro compared to the otherchimeric viruses and rBRSV. Indeed, its growth efficiency was similar tothat of recombinant HRSV (rA2). Thus, the growth of the rBRSV virus wasimproved by replacing the BRSV G and F genes with their HRSVcounterparts (as in rBRSV/A2-GF), and was further improved by placingthe HRSV genes in the promoter-proximal position (as in rBRSV/A2-G1F2),and was yet again further improved by placing the HRSV G and F genes inpositions 3 and 4 (as in rBRSV/A2-G3F4). These results demonstrate howthe properties of viruses within the invention can be systematicallyadjusted by manipulating the origin and order of the viral genes.

[0275] The HEx virus was evaluated in the same way (FIG. 11, bottompanel). Its growth was intermediate to that of rBRSV and rA2, indicatingthat this four-gene replacement retained replication fitness in vitroand indeed exceeded that of its rBRSV parent. It should be noted thatVero cells lack the structural genes for type I interferons, and henceinterferon-specific effects cannot be evaluated. On the other hand, Verocells are a useful substrate for large scale vaccine production, andefficient growth in these cells is an important feature for a vaccinevirus.

[0276] The panel of rBRSV/HRSV chimeric viruses was further evaluatedfor growth on the basis of plaque size in HEp-2 and MDBK cells, theformer of human origin and the latter bovine (FIG. 12, top and bottompanels, respectively). This comparison also included chimeric virusesdescribed in previous examples, namely rBRSV/A2-GF (previously calledrBRSV/A2), rBRSV/A2-MGF, and rBRSV-G1F2. In HEp-2 cells, rHRSV producedlarger plaques than did rBRSV, consistent with the host rangerestriction (FIG. 12, top panel). This discussion first considers thoseviruses in which the NS1 and NS2 genes remained of BRSV origin. In thisgroup, the rBRSV/A2-G3F4, rBRSV/A2-G1F2 and rBRSV/A2-GF viruses producedplaques that were intermediate in size between HRSV and BRSV and whichdecreased in the order given. This is fully consistent with growthkinetic data, and confirms the idea that the introduction of the HRSV Gand F genes into the rBRSV backbone improves its growth in HEp-2 cells,and that further improvement can be obtained by modifying the positionsof these genes. The rBRSV/A2-MGF and rBRSV/A2-MSHGF viruses producedplaques that were smaller than those of rBRSV. While this example showsthat these viruses can be recovered and manipulated, furthercharacterization in vitro and in vivo will be needed to determine thefull characterization of their growth properties.

[0277] Growth in HEp-2 cells was also examined for those viruses inwhich the NS1 and NS2 genes were of HRSV origin. Specifically, pairs ofviruses were compared that were identical except for the origin of theNS1 and NS2 genes. These pairs are listed next, ordered such that thevirus having NS1 and NS2 genes of HRSV is in each pair: rBRSV versusrBRSV/A2-NS1+2; rBRSV/A2-GF versus rBRSV/A2-NS1+2GF; rBRSV/A2-G1F2versus HEx. In each case, the presence of the NS1 and NS2 genes of HRSVorigin provided an increase in plaque size, indicating a modulation ofthe host range restriction. This illustrates how the origin of the NS1and NS2 genes can be selected as a method of predictably modulatinggrowth properties of an HRSV vaccine. In this example, the two geneswere manipulated as a pair, although it is clear that they canalso bemanipulated singly according to the teachings herein.

[0278] The characteristics of these viruses in MDBK bovine cells also isshown in FIG. 12, bottom panel. The host range restriction in thesecells is reversed in comparison with the preceding comparison in HEp-2cells, such that BRSV produced larger plaques than HRSV. The presence ofHRSV G and F genes in the rBRSV backbone did not have much effect ongrowth, while the presence of NS1 and NS2 genes of HRSV originattenuated the virus, presumably because these HRSV-derived interferonantagonists operated less efficiently in bovine cells. Since bovinecells and bovine hosts are not an important target for these candidateHRSV vaccines, these findings with MDBK cells serve mainly to provide aclearer understanding of the functions of these proteins and theircontribution to growth.

[0279] In summary, the foregoing example illustrates how a panel ofrecombinant HRSV vaccine candidates can be readily generated thatexhibit a spectrum of desired growth properties. Clinical evaluation ofselected candidates will provide benchmarks to guide optimization ofvaccine candidates by the methods of this invention. Previous studiesindicated that rBRSV and its rBRSV/A2-GF derivative were over attenuatedin chimpanzees, although the latter virus was an improvement over rBRSV(Buchholz, et al., J. Virol., 74:1187-1199, 2000, incorporated herein byreference). Thus, the further, graded improvements in growth that wereobtained here represent a substantial advance toward optimization of RSVvaccine recombinants.

EXAMPLE VI

[0280] Construction and Recovery of Additional BRSV/HRSV ChimericViruses Containing Substitutions of the N and/or P gene in BRSVBackbones with NS1 and NS2 Proteins of HRSV or BRSV Origin.

[0281] Human parainfluenza virus type 3 (HPIV3) has a bovine counterpart(BPIV3) that exhibits a host range restriction in primates and thusprovides the basis for developing attenuated HPIV3 vaccines based onHPIV3/BPIV3 chimeric viruses. One promising chimera consists of theHPIV3 backbone in which the N ORF was replaced by its BPIV3 counterpart.Remarkably, this chimeric virus replicates efficiently in cell cultureand exhibits an attenuation phenotype in primates (Bailly, et al., J.Virol., 74:3188-95, 2000, incorporated herein by reference).

[0282] Within the present example, investigations were undertaken todetermine whether individual BRSV genes could be replaced by their HRSVcounterparts. Specifically, the N gene and the P gene were individuallysubstituted (FIG. 13A). Additional studies were undertaken to determinewhether two genes could be replaced together. Finally, additionalinvestigations were conducted to determine if gene replacements alsocould be made in a backbone containing the HRSV NS1 and NS2 genes (FIG.13B). It should be noted that these substitutions were made in the rBRSVbackbone, bearing the BRSV G and F genes. In order to make an optimalHRSV vaccine, these would be replaced by their HRSV counterparts,inserted either in the natural gene order positions or in otherpositions, following the teachings set forth herein above whichdemonstrate that such substitutions can be readily made, and indeedgenerally provide improved growth properties.

[0283] A BRSV/HRSV chimera was constructed in which the BRSV N codingsequence was replaced by that of HRSV. This chimera is designatedrBRSV/A2-N (FIG. 13A). For this construction, an Aat II site wasengineered into the rBRSV N gene at nt 2305-2310, which is locatedwithin the last three codons of the N ORF. The substitution was silentat the amino acid level. The same site was engineered into the HRSV NORF of the HRSV antigenomic cDNA. In addition, the HRSV antigenomic cDNAwas modified so that antigenomic nt 1037-1042 in the downstreamnontranslated region of NS2 were changed to a Kpn I site. This Kpn I-AatII HRSV fragment was cloned into the corresponding window of rBRSV,transferring most of the N ORF. The very last few codons of the N ORF ofBRSV and HRSV have the same amino acid coding assignments, and so thefew nt of BRSV N ORF that remain contribute to encode a complete HRSV Nprotein.

[0284] A BRSV/HRSV chimera was constructed in which the BRSV P gene wasreplaced by its HRSV counterpart. This chimera is designated rBRSV/A2-P(FIG. 13A). This employed the above-mentioned Aat II site as well as apreviously-described Mlu I site (see FIG. 9). Transfer of this HRSVfragment to the corresponding window of rBRSV transferred the complete Pgene. As indicated above, the few N ORF nt that were transferred in thisfragment have the same coding assignment in BRSV and HRSV.

[0285] A BRSV/HRSV chimera was constructed in which the above-mentionedN and P sequences of HRSV were transferred to rBRSV, resulting inrBRSV/A2-NP (FIG. 13A). This employed the Kpn I and Mlu I sitesmentioned above.

[0286] The same transfers also were made into a rBRSV backbonecontaining the HRSV NS1 and NS2 genes, namely the rBRSV/A2-NS1+2backbone described in the previous example. This resulted inrBRSV/A2-NS1+2N, rBRSV/A2-NS1+2P, and rBRSV/A2-NS1+2NP (FIG. 13B).

[0287] Each of the foregoing recombinant viruses was readily recoveredfrom cDNA. The rBRSV/A2-P virus replicated in MDBK cells comparably towild-type rBRSV, whereas the replication of the rBRSV/A2-N wasapproximately 10-fold lower and that of the rBRSV/A2-NP virus wasintermediate between these two. Thus, a spectrum of growth propertieswas obtained. Following the methods described above, these viruses canbe modified to bear HRSV G and F genes. In addition, comparable genesubstitutions can be made in the rHRSV backbone. Namely, the HRSV Nand/or P gene can be substituted by the BRSV counterpart. The ability tomake these substitutions in the context of substitutions of the NS1 andNS2 genes offers further flexibility in obtaining an optimal level ofvaccine production in vitro and attenuation and immunogenicity in thehuman vaccinee.

[0288] As indicated by the foregoing examples, genes to be transferredin gene position-shifted RSV can be selected that are likely to interactfunctionally of structurally based on available knowledge of RSVstructure/function. These exchanges and other modifications within geneposition-shifted RSV are further simplified by the fact that proteinsthat interact are juxtaposed in the genome, for example the N and Pnucleocapsid proteins, the M, SH, F and G envelope proteins, and theM2-1, M2-2 and L polymerase components. Thus, additional candidatevaccine strains according to the invention can be achieved, for example,by incorporating two or more juxtaposed genes, e.g., selected from N andP, two or more of the M, SH, F and G envelope genes, or two or more ofthe M2-1, M2-2 and L genes, together as a heterologous insert orsubstitution unit in a recipient or background genome or antigenome.

[0289] For example, the M and SH genes can be replaced together inrBRSV/A2 with their HRSV counterparts. This will result in a virus inwhich the viral envelope proteins (G, F, SH and M) are all of HRSV,while the internal proteins are of BRSV. This can be followed, asneeded, by replacement of additional BRSV genes with their humancounterparts, for example, N and P as another pair, NS1 and NS2 asanother, and M2-1, M2-2 and L as another group. The juxtaposition ofeach pair of genes will simplify the substitutions. At the same time,the converse approach of inserting individual BRSV genes into HRSV,leaving the HRSV G and F antigenic determinants undisturbed, will alsoyield desired vaccine candidates within the invention. For example, oneor more of the N, P, M2-1 and M genes of a human RSV can be individuallyreplaced by their bovine counterparts. Recovered recombinant viruses arethen evaluated for the attenuation phenotype in cell culture, rodents,and nonhuman primates, as exemplified herein. In this manner, theinvention provides for identification of candidate human-bovine chimericRSV vaccine viruses having desired levels of attenuation and protectiveefficacy for treatment and prophylaxis of RSV in various subjects.

EXAMPLE VII

[0290] Improved in vitro Replication of RSV Vaccine Viruses Having aPartial Gene Deletion

[0291] In accordance with the foregoing description, it has been shownthat the efficiency of in vitro replication of RSV is sensitive tochanges in the nucleotide length of the genome. With regard to increasesin length, one type of modification to adjust growth phenotype ofrecombinant RSV can involve the insertion of an additional gene encodinga foreign protein. For example, the coding sequences for bacterialchloramphenicol acetyl transferase (CAT), firefly luciferase, murineinterferon gamma (IFNg), murine interleukin 2 (IL-2), and murinegranulocyte macrophage colony stimulating factor (GM-CSF) have beeninserted individually into the G-F intergenic region. Each of theseinsertions had the effect of reducing the efficiency of virus growth invitro. In one instance, insertion of a CAT transcription cassette ofapproximately 0.76 kb into the G-F intergenic region reduced virusgrowth in vitro 20-fold. The lymphokines were of murine origin and wouldnot be expected to be active in the HEp-2 cells of human origin. Also,the various inserts of comparable size had an effect of comparablemagnitude in reducing RSV growth in vitro. The inhibition reported inthese studies may be attributable to the addition of sequence per se, asopposed to the expression of the various encoded foreign proteins.

[0292] Insertion of a 1.75 kb luciferase cassette into this sameintergenic region had a much greater inhibitory effect on virusreplication (greater than 50-fold reduction), suggesting that largerinserts are more inhibitory. On the other hand, there was some evidencethat this effect also might depend on the location of the insert in thegenome. For example, the insertion of a 0.8 kb transcription cassetteinto the noncoding region of the NS1 gene, placing it in apromoter-proximal position, had only a marginal inhibitory effect onvirus growth (Hallak, et al., J Virol., 74:10508-13, 2000, incorporatedherein by reference). It remains uncertain whether the observed effectwas due to the increase in nucleotide length alone, or due to theaddition of another mRNA-coding unit, or both.

[0293] In other examples, increases in the nt length of recombinant RSVwere made in a single intergenic region. The naturally-occurring RSVintergenic regions that have been analyzed to date range in length from1 to 56 nt. In a recombinant virus lacking the SH gene, the M-SHintergenic region was increased up to 160 nt with a marginal inhibitoryeffect on growth.

[0294] In additional examples, the RSV genome is decreased to yield adesired effect on viral phenotype. In selected embodiments, one or moregenes from the set NS1, NS2, SH, G and M2-2 were deleted singly, or incertain combinations, from recombinant virus without ablating viralinfectivity. Each deletion results in a loss of expression of thedeleted protein, and in most cases resulted in a reduced efficiency ofviral growth in vitro and in vivo. The only exception is that the growthof the SH-deletion virus was not reduced in vitro and, in some celllines, was marginally increased. In another example involving theconstruction of a chimeric virus between RSV strains A2 and B1, theintergenic region between the G and F genes was shortened from 52 nt to5 nt (see, e.g., Whitehead, et al., J. Virol., 73:9773-80, 1999,incorporated herein by reference).

[0295] A number of prior reports have discussed production ofrecombinant RSV with gene or intergenic sequences deleted (Berminghamand Collins, Proc. Natl. Acad. Sci. U.S.A., 96:11259-64, 1999; Bukreyev,et al., J. Virol., 71:8973-82, 1997; Jin, et al., Virology, 273:210-8,2000; Jin, et al., J. Virol., 74:74-82, 2000; Teng and Collins, J.Virol., 73:466-473, 1999; Teng, et al., Journal of Virology, 2000;Whitehead, et al., J. Virol., 73:3438-42, 1999, each incorporated hereinby reference). However, in each case the deletion was accompanied bymodification of open reading frames or other significant genomicfeatures, rendering uncertain the effect of the nucleotide deletion onviral phenotype.

[0296] In the present example, the effect of reducing the length of theRSV genome by deleting sequence from the downstream noncoding region ofthe SH gene is demonstrated. This exemplary partial gene deletion(schematically illustrated in FIG. 14) was constructed using a versionof the antigenome cDNA containing an XmaI site in the G-F intergenicregion, a change which of itself would not be expected to affect theencoded virus. The 141-bp XhoI-PacI window that runs from the end of theSH ORF to the SH gene-end signal was replaced with a synthetic DNAformed from the following two oligonucleotides:TCGAGTtAAtACtTgaTAAAGTAGTTAAT (SEQ ID NO: 7) and TAACTACTTTAtcAaGTaTTaAC(SEQ ID NO: 8) (parts of the XhoI and PacI restriction sites are inbold, nucleotides of the SH open reading frame and termination codon areunderlined, and silent nucleotide changes are indicated in small case).The encoded virus, which was designated RSV/6120, has silent nucleotidesubstitutions in the last three codons and termination codon of the SHORF and has a deletion of 112 nucleotides from the SH downstreamnon-translated region (positions 4499-4610 in the recombinantantigenome) that leaves the gene-end signal intact (Bukreyev, et al., J.Virol. 70:6634-41, 1996, incorporated herein by reference) (FIG. 14).These point mutations and 112-nt deletion thus did not alter the encodedamino acids of any of the viral proteins, did not interrupt any of theknown viral RNA signals, and did not change the number of encoded mRNAs.

[0297] The noncoding changes at the end of the SH gene were made becausethis region is susceptible to instability during growth in bacteria.Indeed, these changes resulted in greatly improved stability inbacteria, a property that is important for the manipulation andpropagation of the antigenome plasmid. Thus, RSV/6120 provided theopportunity to examine the effect of deleting sequence from the genomein the absence of confounding secondary and tertiary effects due toalterations in encoded proteins, RNA signals, or number of encodedmRNAs. It is expected that the five point mutations made in the last fewcodons of the SH ORF will not affect the biological properties of theencoded virus, as evinced by studies of point mutations introduced asmarkers into various genes of recombinant RSV and human and bovineparainfluenza virus type 3 which are not associated with significantchange in biological properties (Collins, et al., Adv. Virus Res.,54:423-51, 1999; Schmidt, et al., J. Virol. 74:8922-9, 2000; Schmidt, etal., J. Virol., 75:4594-603, 2001; Skiadopoulos, et al., J. Virol.,72:1762-8, 1998; Skiadopoulos, et al., J. Virol., 73:1374-81, 1999;Whitehead, et al., J. Virol., 72:4467-4471, 1998; Whitehead, et al., J.Virol., 73:871-7, 1999, each incorporated herein by reference).

[0298] The 6120 virus was analyzed for the efficiency of multi-stepgrowth in parallel with its full-length counterpart, called D53 in threeseparate sets of infections (FIGS. 15A, 15B and 15C). As shown in thefigures, the peak titer of the 6120 virus was reproducibly higher thanthat of the D53 virus by a factor of 1.5- to 2-fold. Thus, themodifications made to the SH gene, in particular the 112-nt noncodingdeletion (representing 0.7% of the genome length), resulted in asubstantial increase in growth efficiency in vitro. Any increase ingrowth efficiency in vitro is an advantage for RSV vaccine production,since the relatively non-robust growth that is characteristic of RSV isan important problem for vaccine development and is anticipated to be acomplication for vaccine production.

[0299] The specific, defined modifications described here provide ageneral tool that can be applied in a variety of contexts to optimizerecombinant vaccine virus growth and other phenotypic characteristics.Based on the present findings, the 15.2 kb genome of RSV provides alarge assemblage of target sites for modification by partial genedeletion or other nucleotide deletions. Typically, changes to beselected in this regard will not involve the 11 viral ORFs and theirtranslation start sites (see, e.g., Kozak, Gene, 234:187-208, 1999,incorporated herein by reference). The viral ORFs account for more than90% of the genome, and thus the typical selection of target sites forpartial deletional modification will be within the remaining,non-translated regions (alternatively referred to as noncoding regions).In addition, target sites for nucleotide deletions in this regard willgenerally exclude cis-acting replication and transcription signals,including the 10-nt gene start and 12- to 13-nt gene end signal thatflank each gene (see, e.g., Collins, et al., Fields Virology,2:1313-1352, 1996, incorporated herein by reference), as well as an11-nt core promoter found at the 3′ end of the genome and the complementof the antigenomic promoter found at the 5′ end of the genome.

[0300] The present example illustrates that, unexpectedly, theefficiency of in vitro growth by RSV can be increased substantially byremoving nontranslated sequence, such as sequence flanking the viralORFs or located between or following genes or in the 3′, and 5′extragenic regions. The example demonstrates that even small deletionsof sequence can yield improved viral growth. This is a highly desiredresult, since the improvement of growth efficiency in vitro facilitateslarge scale vaccine development and production.

Microorganism Deposit Information

[0301] The following materials have been deposited with the AmericanType Culture Collection, 10801 University Boulevard, Manassas, Va.20110-2209, under the conditions of the Budapest Treaty and designatedas follows: Plasmid Accession No. Deposit Date cpts RSV 248 ATCC VR 2450March 22, 1994 cpts RSV 248/404 ATCC VR 2454 March 22, 1994 cpts RSV248/955 ATCC VR 2453 March 22, 1994 cpts RSV 530 ATCC VR 2452 March 22,1994 cpts RSV 530/1009 ATCC VR 2451 March 22, 1994 cpts RSV 530/1030ATCC VR 2455 March 22, 1994 RSV B-1 cp52/2B5 ATCC VR 2542 September 26,1996 RSV B-1 cp-23 ATCC VR 2579 July 15, 1997 p3/7(131) ATCC 97990 April18, 1997 p3/7(131)2G ATCC 97989 April 18, 1997 p218(131) ATCC 97991April 18, 1997

[0302] Although the foregoing invention has been described in detail byway of example for purposes of clarity of understanding, it will beapparent to the artisan that certain changes and modifications may bepractice within the scope of the appended claims which are presented byway of illustration not limitation. In this context, variouspublications and other references have been cited within the foregoingdisclosure for economy of description. Each of these references areincorporated herein by reference in its entirety for all purposes.

1 23 1 6 DNA Artificial Sequence Description of Artificial SequenceArtificial Respiratory Syncytial Virus 1 catatt 6 2 6 DNA ArtificialSequence Description of Artificial Sequence Artificial RespiratorySyncytial Virus 2 cacaat 6 3 25 DNA Artificial Sequence Description ofArtificial Sequence Artificial Respiratory Syncytial Virus 3 ttaattaaaaacatattatc acaaa 25 4 13 DNA Artificial Sequence Description ofArtificial Sequence Artificial Respiratory Syncytial Virus 4 cacaattgcatgc 13 5 18 DNA Artificial Sequence Description of Artificial SequenceArtificial Respiratory Syncytial Virus 5 ttaattaaaa acacaatt 18 6 64 DNAArtificial Sequence Description of Artificial Sequence ArtificialRespiratory Syncytial Virus 6 ttaaacttaa aaatggttta tgtcgaggaataaaatcgat taacaaccaa tcattcaaaa 60 agat 64 7 29 DNA Artificial SequenceDescription of Artificial Sequence Artificial Respiratory SyncytialVirus 7 tcgagttaat acttgataaa gtagttaat 29 8 23 DNA Artificial SequenceDescription of Artificial Sequence Artificial Respiratory SyncytialVirus 8 taactacttt atcaagtatt aac 23 9 57 DNA Artificial SequenceDescription of Artificial Sequence Artificial Respiratory SyncytialVirus 9 ggggcaaata agaatttgat aagtaccact taaatttaac tcccttgctt agcgatg57 10 9 DNA Artificial Sequence Description of Artificial SequenceArtificial Respiratory Syncytial Virus 10 ttagcgatg 9 11 19 DNAArtificial Sequence Description of Artificial Sequence ArtificialRespiratory Syncytial Virus 11 catattgggg caaataagc 19 12 19 DNAArtificial Sequence Description of Artificial Sequence ArtificialRespiratory Syncytial Virus 12 cacaatgggg caaataagc 19 13 55 DNAArtificial Sequence Description of Artificial Sequence ArtificialRespiratory Syncytial Virus 13 ggggcaaata caagttaatt cgcggccgccccctctcttc tttctacaga aaatg 55 14 35 DNA Artificial Sequence Descriptionof Artificial Sequence Artificial Respiratory Syncytial Virus 14gcggccgcta aatttaactc ccttgcttag cgatg 35 15 31 DNA Artificial SequenceDescription of Artificial Sequence Artificial Respiratory SyncytialVirus 15 cacaatgggg caaaataagc ttagcggccg c 31 16 5 DNA ArtificialSequence Description of Artificial Sequence Artificial RespiratorySyncytial Virus 16 taaaa 5 17 12 DNA Artificial Sequence Description ofArtificial Sequence Artificial Respiratory Syncytial Virus 17 taaagacgcgtt 12 18 24 DNA Artificial Sequence Description of Artificial SequenceArtificial Respiratory Syncytial Virus 18 agttagtaaa aataaagacg cgtt 2419 29 DNA Artificial Sequence Description of Artificial SequenceArtificial Respiratory Syncytial Virus 19 ttatgtcgac tggggcaaatgcaaacatg 29 20 6 PRT Artificial Sequence Description of ArtificialSequence Artificial Respiratory Syncytial Virus 20 Arg Ala Arg Val AsnThr 1 5 21 23 DNA Artificial Sequence Description of Artificial SequenceArtificial Respiratory Syncytial Virus 21 agagctcgag tcaacacata gca 2322 24 DNA Artificial Sequence Description of Artificial SequenceArtificial Respiratory Syncytial Virus 22 tataaagtag ttaattaaaa atag 2423 43 DNA Artificial Sequence Description of Artificial SequenceArtificial Respiratory Syncytial Virus 23 agagctcgag ttaatacttgataaagtagt taattaaaaa tag 43

What is claimed is:
 1. An isolated infectious recombinant respiratorysyncytial virus (RSV) comprising a major nucleocapsid (N) protein, anucleocapsid phosphoprotein (P), a large polymerase protein (L), a RNApolymerase elongation factor, and a partial or complete recombinant RSVgenome or antigenome having one or more shifted RSV gene(s) or genomesegment(s) within said recombinant genome or antigenome that is/arepositionally shifted to a more promoter-proximal or promoter-distalposition relative to a position of said RSV gene(s) or genome segment(s)within a wild type RSV genome or antigenome.
 2. The isolated infectiousrecombinant RSV of claim 1, wherein said one or more shifted gene(s) orgenome segment(s) is/are shifted to a more promoter-proximal orpromoter-distal position by deletion or insertion of one or moredisplacement polynucleotide(s) within said partial or completerecombinant RSV genome or antigenome.
 3. The isolated infectiousrecombinant RSV of claim 2, wherein said displacement polynucleotide(s)comprise(s) one or more polynucleotide insert(s) of between 150nucleotides (nts) and 4,000 nucleotides in length which is inserted in anon-coding region (NCR) of the genome or antigenome or as a separategene unit (GU), said polynucleotide insert lacking a complete openreading frame (ORF) and specifying an attenuated phenotype in saidrecombinant RSV.
 4. The isolated infectious recombinant RSV of claim 3,wherein said polynucleotide insert(s) comprises one or more RSV gene(s)or genome segment(s).
 5. The isolated infectious recombinant RSV ofclaim 2, wherein said displacement polynucleotide(s) comprise(s) one ormore RSV gene(s) or genome segment(s) selected from RSV NS1, NS2, N, P,M, SH, M2(ORF1), M2(ORF2), L, F and G genes and genome segments andleader, trailer and intergenic regions of the RSV genome and segmentsthereof.
 6. The isolated infectious recombinant RSV of claim 2, whereinsaid displacement polynucleotide(s) comprise(s) one or more bovine RSV(BRSV) or human RSV (HRSV) gene(s) or genome segment(s) selected fromRSV NS1, NS2, N, P, M, SH, M2(ORF1), M2(ORF2), L, F and G gene(s) orgenome segment(s) and leader, trailer and intergenic regions of the RSVgenome or segments thereof.
 7. The isolated infectious recombinant RSVof claim 6, wherein said displacement polynucleotide(s) is/are deletedto form the recombinant RSV genome or antigenome to cause a positionalshift of said one or more shifted RSV gene(s) or genome segment(s)within said recombinant genome or antigenome to a more promoter-proximalposition relative to a position of said RSV gene(s) or genome segment(s)within a wild type RSV genome or antigenome.
 8. The isolated infectiousrecombinant RSV of claim 7, wherein said displacement polynucleotide(s)that is/are deleted to form the recombinant RSV genome or antigenomecomprise one or more RSV NS1, NS2, SH, M2(ORF2), or G gene(s) or genomesegment(s) thereof.
 9. The isolated infectious recombinant RSV of claim8, wherein a displacement polynucleotide comprising a RSV NS1 gene isdeleted to form the recombinant RSV genome or antigenome.
 10. Theisolated infectious recombinant RSV of claim 8, wherein a displacementpolynucleotide comprising a RSV NS2 gene is deleted to form therecombinant RSV genome or antigenome.
 11. The isolated infectiousrecombinant RSV of claim 8, wherein a displacement polynucleotidecomprising a RSV SH gene is deleted to form the recombinant RSV genomeor antigenome.
 12. The isolated infectious recombinant RSV of claim 8,wherein a displacement polynucleotide comprising RSV M2(ORF2) is deletedto form the recombinant RSV genome or antigenome.
 13. The isolatedinfectious recombinant RSV of claim 8, wherein a displacementpolynucleotide comprising a RSV G gene is deleted to form therecombinant RSV genome or antigenome or antigenome.
 14. The isolatedinfectious recombinant RSV of claim 8, wherein the RSV F and G genes areboth deleted to form the recombinant RSV genome or antigenome orantigenome.
 15. The isolated infectious recombinant RSV of claim 8,wherein the RSV NS1 and NS2 genes are both deleted to form therecombinant RSV genome or antigenome or antigenome.
 16. The isolatedinfectious recombinant RSV of claim 8, wherein the RSV SH and NS2 genesare both deleted to form the recombinant RSV genome or antigenome orantigenome.
 17. The isolated infectious recombinant RSV of claim 8,wherein the RSV SH, NS1 and NS2 genes are all deleted to form therecombinant RSV genome or antigenome or antigenome.
 18. The isolatedinfectious recombinant RSV of claim 7, wherein said displacementpolynucleotide(s) comprise(s) one or more deletion(s) within anontranslated sequence at the beginning or end of an RSV open readingframe or in an intergenic region or 3′ leader or 5′ trailer portion ofthe RSV genome.
 19. The isolated infectious recombinant RSV of claim 18,wherein said displacement polynucleotides comprise a partial genedeletion.
 20. The isolated infectious recombinant RSV of claim 19,wherein said partial gene deletion is a partial deletion of the SH gene.21. The isolated infectious recombinant RSV of claim 20, wherein saidpartial deletion of the SH gene comprises a deletion within the SHdownstream non-translated region.
 22. The isolated infectiousrecombinant RSV of claim 21, which is RSV 6120 having a deletion of 112nucleotides at positions 4499-4610 in the recombinant RSV antigenome.23. The isolated infectious recombinant RSV of claim 7, wherein saiddisplacement polynucleotide(s) is/are selected from one or moreregion(s) of a downstream untranslated sequence of an RSV gene.
 24. Theisolated infectious recombinant RSV of claim 23, wherein said downstreamuntranslated sequence(s) is/are from NS1 (positions 519-563), NS2(positions 1003-1086), P (positions 3073-3230), M (positions 4033-4197),F(positions 7387-7539), and/or M2 (positions 8433-8490) genes.
 25. Theisolated infectious recombinant RSV of claim 7, wherein saiddisplacement polynucleotide(s) is/are selected from one or moreregion(s) of a upstream untranslated sequence of an RSV gene.
 26. Theisolated infectious recombinant RSV of claim 25, wherein said one ormore upstream untranslated sequences is/are from NS1 (positions 55-96),NS2 (positions 606-624) and/or SH (positions 4231-4300).
 27. Theisolated infectious recombinant RSV of claim 7, wherein saiddisplacement polynucleotide comprises a deletion of nucleotides 4683 to4685 of the RSV G gene.
 28. The isolated infectious recombinant RSV ofclaim 7, wherein said displacement polynucleotide(s) is/are selectedfrom one or more RSV intergenic sequences.
 29. The isolated infectiousrecombinant RSV of claim 7, wherein said displacement polynucleotide(s)is/are selected from nucleotides within the RSV 5′ trailer region. 30.The isolated infectious recombinant RSV of claim 29, wherein a portionof the 5′ trailer region that immediately follows the L gene is reducedin size by 75 nucleotides, 100 nucleotides, 125 nucleotides, or more,leaving intact the 5′ genomic terminus.
 31. The isolated infectiousrecombinant RSV of claim 7, wherein said displacement polynucleotide(s)is/are selected from nucleotides within the RSV 3′ leader region. 32.The isolated infectious recombinant RSV of claim 31, wherein a portionof the 3′ trailer region that excludes a core portion of the viralpromoter located within the first 11 nucleotides of the 3′ leader isdeleted.
 33. The isolated infectious recombinant RSV of claim 7, whereina partial or complete deletion from one or any combination of the RSVNS1, NS2, SH, F and/or M2 genes yields an adjustable reduction in genomelength of between 1-806 nucleotides.
 34. The isolated infectiousrecombinant RSV of claim 7, wherein a partial or complete deletion fromone or any combination of RSV intergenic regions yields an adjustablereduction in genome length of between 1-198 nucleotides.
 35. Theisolated infectious recombinant RSV of claim 7, wherein a partial orcomplete deletion from one or any combination of RSV intergenic regionsyields an adjustable reduction in genome length of between 1-198nucleotides.
 36. The isolated infectious recombinant RSV of claim 6,wherein said displacement polynucleotide(s) is/are added, substituted,or rearranged within the recombinant RSV genome or antigenome to cause apositional shift of said one or more shifted RSV gene(s) or genomesegment(s) within said recombinant genome or antigenome to a morepromoter-proximal or promoter-distal position relative to a position ofsaid RSV gene(s) or genome segment(s) within a wild type RSV genome orantigenome.
 37. The isolated infectious recombinant RSV of claim 36,wherein said displacement polynucleotide(s) added, substituted, orrearranged within the recombinant RSV genome or antigenome comprise(s)one or more RSV NS1, NS2, SH, M2(ORF2), F, and/or G gene(s) or genomesegment(s) thereof.
 38. The isolated infectious recombinant RSV of claim36, wherein said displacement polynucleotide(s) comprise(s) one or moreRSV gene(s) or genome segment(s) encoding one or more RSVglycoprotein(s) or immunogenic domain(s) or epitope(s) thereof.
 39. Theisolated infectious recombinant RSV of claim 38, wherein saiddisplacement polynucleotide(s) is/are selected from gene(s) or genomesegment(s) encoding RSV F, G, and/or SH glycoprotein(s) or immunogenicdomain(s) or epitope(s) thereof.
 40. The isolated infectious recombinantRSV of claim 1, wherein one or more RSV glycoprotein gene(s) or genomesegments of RSV F, G and SH is/are added, substituted or rearrangedwithin said recombinant RSV genome or antigenome to a position that ismore promoter-proximal compared to a wild type gene order position ofsaid one or more RSV glycoprotein gene(s).
 41. The isolated infectiousrecombinant RSV of claim 40, wherein the RSV glycoprotein gene G isrearranged within said recombinant RSV genome or antigenome to a geneorder position that is more promoter-proximal compared to the wild typegene order position of G.
 42. The isolated infectious recombinant RSV ofclaim 41, wherein the RSV glycoprotein gene G is shifted to gene orderposition 1 within said recombinant RSV genome or antigenome.
 43. Theisolated infectious recombinant RSV of claim 40, wherein the RSVglycoprotein gene F is rearranged within said recombinant RSV genome orantigenome to a gene order position that is more promoter-proximalcompared to the wild type gene order position of F.
 44. The isolatedinfectious recombinant RSV of claim 43, wherein the RSV glycoproteingene F is shifted to gene order position 1 within said recombinant RSVgenome or antigenome.
 45. The isolated infectious recombinant RSV ofclaim 40, wherein both RSV glycoprotein genes G and F are rearrangedwithin said recombinant RSV genome or antigenome to gene order positionsthat are more promoter-proximal compared to the wild type gene orderpositions of G and F.
 46. The isolated infectious recombinant RSV ofclaim 45, wherein the RSV glycoprotein gene G is shifted to gene orderposition 1 and the RSV glycoprotein gene F is shifted to gene orderposition 2 within said recombinant RSV genome or antigenome.
 47. Theisolated infectious recombinant RSV of claim 40, wherein one or more RSVNS1, NS2, SH, M2(ORF2), or G gene(s) or genome segment(s) thereof is/aredeleted in the recombinant RSV genome or antigenome.
 48. The isolatedinfectious recombinant RSV of claim 40, wherein a displacementpolynucleotide comprising a RSV NS1 gene is deleted to form therecombinant RSV genome or antigenome.
 49. The isolated infectiousrecombinant RSV of claim 40, wherein a displacement polynucleotidecomprising a RSV NS2 gene is deleted to form the recombinant RSV genomeor antigenome.
 50. The isolated infectious recombinant RSV of claim 40wherein a displacement polynucleotide comprising a RSV SH gene isdeleted to form the recombinant RSV genome or antigenome.
 51. Theisolated infectious recombinant RSV of claim 50, wherein the RSVglycoprotein gene G is rearranged within said recombinant RSV genome orantigenome to a gene order position that is more promoter-proximalcompared to the wild type gene order position of G.
 52. The isolatedinfectious recombinant RSV of claim 51, wherein the RSV glycoproteingene G is shifted to gene order position 1 within said recombinant RSVgenome or antigenome.
 53. The isolated infectious recombinant RSV ofclaim 52, which is G1/ΔSH.
 54. The isolated infectious recombinant RSVof claim 50, wherein the RSV glycoprotein gene F is rearranged withinsaid recombinant RSV genome or antigenome to a gene order position thatis more promoter-proximal compared to the wild type gene order positionof F.
 55. The isolated infectious recombinant RSV of claim 54, whereinthe RSV glycoprotein gene F is shifted to gene order position 1 withinsaid recombinant RSV genome or antigenome.
 56. The isolated infectiousrecombinant RSV of claim 55, which is F1/ΔSH.
 57. The isolatedinfectious recombinant RSV of claim 50, wherein both RSV glycoproteingenes G and F are rearranged within said recombinant RSV genome orantigenome to gene order positions that are more promoter-proximalcompared to the wild type gene order positions of G and F.
 58. Theisolated infectious recombinant RSV of claim 57, wherein the RSVglycoprotein gene G is shifted to gene order position 1 and the RSVglycoprotein gene F is shifted to gene order position 2 within saidrecombinant RSV genome or antigenome.
 59. The isolated infectiousrecombinant RSV of claim 58, which is G1F2/ΔSH.
 60. The isolatedinfectious recombinant RSV of claim 40, wherein the RSV SH and NS2 genesare both deleted to form the recombinant RSV genome or antigenome orantigenome.
 61. The isolated infectious recombinant RSV of claim 60,wherein both RSV glycoprotein genes G and F are rearranged within saidrecombinant RSV genome or antigenome to gene order positions that aremore promoter-proximal compared to the wild type gene order positions ofG and F.
 62. The isolated infectious recombinant RSV of claim 61,wherein the RSV glycoprotein gene G is shifted to gene order position 1and the RSV glycoprotein gene F is shifted to gene order position 2within said recombinant RSV genome or antigenome.
 63. The isolatedinfectious recombinant RSV of claim 62, which is G1F2/ΔNS2ΔSH.
 64. Theisolated infectious recombinant RSV of claim 40, wherein the RSV SH, NS1and NS2 genes are all deleted to form the recombinant RSV genome orantigenome or antigenome.
 65. The isolated infectious recombinant RSV ofclaim 64, wherein both RSV glycoprotein genes G and F are rearrangedwithin said recombinant RSV genome or antigenome to gene order positionsthat are more promoter-proximal compared to the wild type gene orderpositions of G and F.
 66. The isolated infectious recombinant RSV ofclaim 65, wherein the RSV glycoprotein gene G is shifted to gene orderposition 1 and the RSV glycoprotein gene F is shifted to gene orderposition 2 within said recombinant RSV genome or antigenome.
 67. Theisolated infectious recombinant RSV of claim 66, which isG1F2/ΔNS2ΔNS2ΔSH.
 68. The isolated infectious recombinant RSV of claim1, wherein the recombinant genome or antigenome comprises a partial orcomplete human RSV (HRSV) or bovine RSV (BRSV) background genome orantigenome combined with one or more heterologous gene(s) or genomesegment(s) from a different RSV to form a human-bovine chimeric RSVgenome or antigenome.
 69. The isolated infectious recombinant RSV ofclaim 68, wherein the heterologous gene or genome segment is added orsubstituted at a position that is more promoter-proximal orpromoter-distal compared to a wild type gene order position of acounterpart gene or genome segment within the partial or complete HRSVor BRSV background genome or antigenome.
 70. The isolated infectiousrecombinant RSV of claim 69, wherein both human RSV glycoprotein genes Gand F are substituted at gene order positions 1 and 2, respectively, toreplace counterpart G and F glycoprotein genes deleted at wild typepositions 7 and 8, respectively in a partial bovine RSV backgroundgenome or antigenome.
 71. The isolated infectious recombinant RSV ofclaim 70, which is rBRSV/A2-G1F2.
 72. The isolated infectiousrecombinant RSV of claim 69, wherein one or more human RSVnon-structural and/or envelope-associated genes selected from NS1, NS2,F, G, SH, and M is/are added or substituted within a partial or completebovine RSV background genome or antigenome.
 73. The isolated infectiousrecombinant RSV of claim 69, wherein one or more human RSVenvelope-associated genes selected from F, G, SH, and M is/are added orsubstituted within a partial bovine RSV background genome or antigenomein which one or more envelope-associated genes selected from F, G, SH,and M is/are deleted.
 74. The isolated infections recombinant RSV ofclaim 73, wherein human RSV envelope-associated genes F, G, and M areadded within a partial bovine RSV background genome or antigenome inwhich all of the envelope-associated genes F, G, SH, and M are deleted.75. The isolated infectious recombinant RSV of claim 74, which isrBRSV/A2-MGF.
 76. The isolated infectious recombinant RSV of claim 69,wherein both human RSV glycoprotein genes G and F are substituted atgene order positions 3 and 4, respectively, to replace counterpart G andF glycoprotein genes deleted at wild type positions 7 and 8,respectively in a partial bovine RSV background genome or antigenome.77. The isolated infectious recombinant RSV of claim 76, which isrBRSV/A2-G3F4.
 78. The isolated infectious recombinant RSV of claim 69,wherein both human RSV glycoprotein genes G and F are substituted atgene order positions 1 and 2, respectively, to replace counterpart G andF glycoprotein genes deleted at wild type positions 7 and 8,respectively, and wherein human RSV genes NS1 and NS2 are subsituted fortheir bovine counterpart genes, in a partial bovine RSV backgroundgenome or antigenome.
 79. The isolated infectious recombinant RSV ofclaim 78, which is rBRSV/A2-G1F2NS3NS4.
 80. The isolated infectiousrecombinant RSV of claim 1, in which RSV M2(ORF1) is shifted to a morepromoter-proximal position within the recombinant RSV genome orantigenome to upregulate transcription of the recombinant virus.
 81. Theisolated infectious recombinant RSV of claim 1, wherein the recombinantgenome or antigenome incorporates at least one and up to a fullcomplement of attenuating mutations present within a panel of mutanthuman RSV strains, said panel comprising cpts RSV 248 (ATCC VR 2450),cpts RSV 248/404 (ATCC VR 2454), cpts RSV 248/955 (ATCC VR 2453), cptsRSV 530 (ATCC VR 2452), cpts RSV 530/1009 (ATCC VR 2451), cpts RSV530/1030 (ATCC VR 2455), RSV B-1 cp52/2B5 (ATCC VR 2542), and RSV B-1cp-23 (ATCC VR 2579).
 82. The isolated infectious recombinant RSV ofclaim 81, wherein the recombinant genome or antigenome incorporatesattenuating mutations adopted from different mutant RSV strains.
 83. Theisolated infectious recombinant RSV of claim 1, wherein the recombinantgenome or antigenome incorporates at least one and up to a fullcomplement of attenuating mutations specifying an amino acidsubstitution at Val267 in the RSV N gene, Glu218 and/or Thr523 in theRSV F gene, Asn43, Cys319, Phe 521, Gln831, Met1169, Tyr1321 and/or His1690 in the RSV polymerase gene L, and a nucleotide substitution in thegene-start sequence of gene M2.
 84. The isolated infectious recombinantRSV of claim 83, wherein the recombinant genome or antigenomeincorporates at least two attenuating mutations.
 85. The isolatedinfectious recombinant RSV of claim 83, wherein the recombinant genomeor antigenome includes at least one attenuating mutation stabilized bymultiple nucleotide changes in a codon specifying the mutation.
 86. Theisolated infectious recombinant RSV of claim 1, wherein the recombinantgenome or antigenome further comprises a nucleotide modificationspecifying a phenotypic change selected from a change in growthcharacteristics, attenuation, temperature-sensitivity, cold-adaptation,plaque size, host-range restriction, or a change in immunogenicity. 87.The isolated infectious recombinant RSV of claim 86, wherein thenucleotide modification alters a SH, NS1, NS2, M2ORF2, or G gene of therecombinant virus.
 88. The isolated infectious recombinant RSV of claim87, wherein a SH, NS1, NS2, M2 ORF2, or G gene of the recombinant virusis deleted in whole or in part or expression of the gene is ablated byintroduction of one or more stop codons in an open reading frame of thegene.
 89. The isolated infectious recombinant RSV of claim 86, whereinthe nucleotide modification comprises a nucleotide deletion, insertion,substitution, addition or rearrangement of a cis-acting regulatorysequence of a selected gene within the recombinant RSV genome orantigenome.
 90. The isolated infectious recombinant RSV of claim 89,wherein a gene end (GE) signal of the NS1 or NS2 gene is modified. 91.The isolated infectious recombinant RSV of claim 89, wherein thenucleotide modification comprises an insertion, deletion, substitution,or rearrangement of a translational start site within the recombinantgenome or antigenome.
 92. The isolated infectious recombinant RSV ofclaim 91, wherein the translational start site for a secreted form ofthe RSV G glycoprotein is ablated.
 93. The isolated infectiousrecombinant RSV of claim 1, wherein the recombinant genome or antigenomeis modified to encode a non-RSV molecule selected from a cytokine, aT-helper epitope, a restriction site marker, or a protein of a microbialpathogen capable of eliciting a protective immune response against saidpathogen in a mammalian host.
 94. The isolated infectious recombinantRSV of claim 93, which incorporates one or more gene(s) and/or genomesegment(s) from parainfluenza virus (PIV).
 95. The isolated infectiousrecombinant RSV of claim 94, wherein the recombinant genome orantigenome encodes a HN or F glycoprotein, or an ectodomain orimmunogenic epitope of HN or F, of PIV1, PIV2, or PIV3.
 96. The isolatedinfectious recombinant RSV of claim 1 which is a virus.
 97. The isolatedinfectious recombinant RSV of claim 1 which is a subviral particle. 98.The isolated infectious recombinant RSV of claim 2, wherein saiddisplacement polynucleotide is added within or deleted from a noncodingregion of the recombinant RSV genome or antigenome.
 99. The isolatedinfectious recombinant RSV of claim 1, wherein the recombinant genome orantigenome incorporates antigenic determinants from one or both subgroupA and subgroup B human RSV.
 100. A method for stimulating the immunesystem of an individual to induce protection against RSV which comprisesadministering to the individual an immunologically sufficient amount ofthe recombinant RSV of claim 1 combined with a physiologicallyacceptable carrier.
 101. The method of claim 100, wherein therecombinant RSV is administered in a dose of 10³ to 10⁶ PFU.
 102. Themethod of claim 100, wherein the recombinant RSV is administered to theupper respiratory tract.
 103. The method of claim 100, wherein therecombinant RSV is administered by spray, droplet or aerosol.
 104. Themethod of claim 100, wherein the recombinant RSV is administered to anindividual seronegative for antibodies to RSV or possessingtransplacentally acquired maternal antibodies to RSV.
 105. The method ofclaim 100, wherein the recombinant RSV elicits an immune responseagainst either human RSV A or RSV B.
 106. The method of claim 100,wherein the recombinant RSV elicits an immune response against bothhuman RSV A and RSV B.
 107. The method of claim 100, wherein therecombinant RSV elicits an immune response against either human RSV A orRSV B and is co-administered with an immunologically sufficient amountof a second attenuated RSV capable of eliciting an immune responseagainst human RSV A or RSV B, whereby an immune response is elicitedagainst both human RSV A and RSV B.
 108. The method of claim 107,wherein the recombinant RSV and second attenuated RSV are administeredsimultaneously as a mixture.
 109. An immunogenic composition to elicitan immune response against RSV comprising an immunologically sufficientamount of the recombinant RSV of claim 1 in a physiologically acceptablecarrier.
 110. The immunogenic composition of claim 109, formulated in adose of 10³ to 10⁶ PFU.
 111. The immunogenic composition of claim 109,formulated for administration to the upper respiratory tract by spray,droplet or aerosol.
 112. The immunogenic composition of claim 109,wherein the recombinant RSV elicits an immune response against eitherhuman RSV A or RSV B or both human RSV A and RSV B.
 113. The isolatedinfectious recombinant RSV of claim 1, wherein the recombinant genome orantigenome comprises a partial or complete RSV vector genome orantigenome combined with one or more heterologous genes or genomesegments encoding one or more antigenic determinants of one or moreheterologous pathogens.
 114. The isolated infectious recombinant RSV ofclaim 113, wherein said one or more heterologous pathogens is aheterologous RSV and said heterologous gene(s) or genome segment(s)encode(s) one or more RSV NS1, NS2, N, P, M, SH, M2(ORF1), M2(ORF2), L,F or G protein(s) or fragment(s) thereof.
 115. The isolated infectiousrecombinant RSV of claim 113, wherein the vector genome or antigenome isa partial or complete RSV A genome or antigenome and the heterologousgene(s) or genome segment(s) encoding the antigenic determinant(s)is/are of a RSV B subgroup virus.
 116. The isolated infectiousrecombinant RSV of claim 113, wherein the chimeric genome or antigenomeincorporates one or more gene(s) or genome segment(s) of a BRSV thatspecifies attenuation.
 117. The isolated infectious recombinant RSV ofclaim 113, wherein one or more HPIV1, HPIV2, or HPIV3 gene(s) or genomesegment(s) encoding one or more HN and/or F glycoprotein(s) or antigenicdomain(s), fragment(s) or epitope(s) thereof is/are added to orincorporated within the partial or complete HRSV vector genome orantigenome.
 118. The isolated infectious recombinant RSV of claim 113,wherein the vector genome or antigenome is a partial or complete BRSVgenome or antigenome and the heterologous gene(s) or genome segment(s)encoding the antigenic determinant(s) is/are of one or more HRSV(s).119. The isolated infectious recombinant RSV of claim 118, wherein thepartial or complete BRSV genome or antigenome incorporates one or moregene(s) or genome segment(s) encoding one or more HRSV glycoproteingenes selected from F, G and SH, or one or more genome segment(s)encoding cytoplasmic domain, transmembrane domain, ectodomain orimmunogenic epitope portion(s) of F, G, and/or SH of HRSV.
 120. Theisolated infectious recombinant RSV of claim 113, wherein the vectorgenome or antigenome is a partial or complete HRSV or BRSV genome orantigenome and the heterologous pathogen is selected from measles virus,subgroup A and subgroup B respiratory syncytial viruses, mumps virus,human papilloma viruses, type 1 and type 2 human immunodeficiencyviruses, herpes simplex viruses, cytomegalovirus, rabies virus, EpsteinBarr virus, filoviruses, bunyaviruses, flaviviruses, alphaviruses andinfluenza viruses.
 121. The isolated infectious recombinant RSV of claim120, wherein said one or more heterologous antigenic determinant(s)is/are selected from measles virus HA and F proteins, subgroup A orsubgroup B respiratory syncytial virus F, G, SH and M2 proteins, mumpsvirus HN and F proteins, human papilloma virus L1 protein, type 1 ortype 2 human immunodeficiency virus gp160 protein, herpes simplex virusand cytomegalovirus gB, gC, gD, gE, gG, gH, gI, gJ, gK, gL, and gMproteins, rabies virus G protein, Epstein Barr Virus gp350 protein;filovirus G protein, bunyavirus G protein, Flavivirus E and NS1proteins, and alphavirus E protein, and antigenic domains, fragments andepitopes thereof.
 122. The isolated infectious recombinant RSV of claim121, wherein the heterologous pathogen is measles virus and theheterologous antigenic determinant(s) is/are selected from the measlesvirus HA and F proteins and antigenic domains, fragments and epitopesthereof.
 123. The isolated infectious recombinant RSV of claim 122,wherein a transcription unit comprising an open reading frame (ORF) of ameasles virus HA gene is added to or incorporated within a HRSV vectorgenome or antigenome.
 124. An isolated polynucleotide moleculecomprising a recombinant RSV genome or antigenome having one or moreshifted RSV gene(s) or genome segment(s) within said recombinant genomeor antigenome that is/are positionally shifted to a morepromoter-proximal or promoter-distal position relative to a position ofsaid RSV gene(s) or genome segment(s) within a wild type RSV genome orantigenome.
 125. The isolated polynucleotide molecule of claim 124,wherein said one or more shifted gene(s) or genome segment(s) is/areshifted to a more promoter-proximal position by insertion or deletion ofone or more displacement polynucleotide(s) within said partial orcomplete recombinant RSV genome or antigenome.
 126. The isolatedpolynucleotide molecule of claim 125, wherein said displacementpolynucleotide(s) comprise(s) one or more polynucleotide insert(s) ofbetween 150 nucleotides (nts) and 4,000 nucleotides in length which isinserted in a non-coding region (NCR) of the genome or antigenome or asa separate gene unit (GU), said polynucleotide insert lacking a completeopen reading frame (ORF) and specifying an attenuated phenotype in saidrecombinant RSV.
 127. The isolated polynucleotide molecule of claim 126,wherein said polynucleotide insert(s) comprises one or more RSV gene(s)or genome segment(s).
 128. The isolated polynucleotide molecule of claim127, wherein said displacement polynucleotide(s) comprise(s) one or morebovine RSV (BRSV) or human RSV (HRSV) gene(s) or genome segment(s)selected from RSV NS1, NS2, N, P, M, SH, M2(ORF1), M2(ORF2), L, F and Ggene(s) or genome segment(s) and leader, trailer and intergenic regionsof the RSV genome or segments thereof.
 129. The isolated polynucleotidemolecule of claim 128, wherein said displacement polynucleotide(s)is/are deleted to form the recombinant RSV genome or antigenome to causea positional shift of said one or more shifted RSV gene(s) or genomesegment(s) within said recombinant genome or antigenome to a morepromoter-proximal position relative to a position of said RSV gene(s) orgenome segment(s) within a wild type RSV genome or antigenome.
 130. Theisolated polynucleotide molecule of claim 129, wherein said displacementpolynucleotide(s) that is/are deleted to form the recombinant RSV genomeor antigenome comprise one or more RSV NS1, NS2, SH, M2(ORF2), or Ggene(s) or genome segment(s) thereof.
 131. The isolated polynucleotidemolecule of claim 130, wherein a displacement polynucleotide comprisinga RSV NS1 gene is deleted to form the recombinant RSV genome orantigenome.
 132. The isolated polynucleotide molecule of claim 130,wherein a displacement polynucleotide comprising a RSV NS2 gene isdeleted to form the recombinant RSV genome or antigenome.
 133. Theisolated polynucleotide molecule of claim 130, wherein a displacementpolynucleotide comprising a RSV SH gene is deleted to form therecombinant RSV genome or antigenome.
 134. The isolated polynucleotidemolecule of claim 130, wherein a displacement polynucleotide comprisingRSV M2(ORF2) is deleted to form the recombinant RSV genome orantigenome.
 135. The isolated polynucleotide molecule of claim 130,wherein a displacement polynucleotide comprising a RSV G gene is deletedto form the recombinant RSV genome or antigenome or antigenome.
 136. Theisolated polynucleotide molecule of claim 130, wherein the RSV F and Ggenes are both deleted to form the recombinant RSV genome or antigenomeor antigenome.
 137. The isolated polynucleotide molecule of claim 130,wherein the RSV NS1 and NS2 genes are both deleted to form therecombinant RSV genome or antigenome or antigenome.
 138. The isolatedpolynucleotide molecule of claim 130, wherein the RSV SH and NS2 genesare both deleted to form the recombinant RSV genome or antigenome orantigenome.
 139. The isolated polynucleotide molecule of claim 130,wherein the RSV SH, NS1 and NS2 genes are all deleted to form therecombinant RSV genome or antigenome or antigenome.
 140. The isolatedpolynucleotide molecule of claim 128, wherein said displacementpolynucleotide(s) is/are added, substituted, or rearranged within therecombinant RSV genome or antigenome to cause a positional shift of saidone or more shifted RSV gene(s) or genome segment(s) within saidrecombinant genome or antigenome to a more promoter-proximal orpromoter-distal position relative to a position of said RSV gene(s) orgenome segment(s) within a wild type RSV genome or antigenome.
 141. Theisolated polynucleotide molecule of claim 140, wherein said displacementpolynucleotide(s) added, substituted, or rearranged within therecombinant RSV genome or antigenome comprise(s) one or more RSV NS1,NS2, SH, M2(ORF2), F, and/or G gene(s) or genome segment(s) thereof.142. The isolated polynucleotide molecule of claim 140, wherein saiddisplacement polynucleotide(s) comprise(s) one or more RSV gene(s) orgenome segment(s) encoding one or more RSV glycoprotein(s) orimmunogenic domain(s) or epitope(s) thereof.
 143. The isolatedpolynucleotide molecule of claim 141, wherein said displacementpolynucleotide(s) is/are selected from gene(s) or genome segment(s)encoding RSV F, G, and/or SH glycoprotein(s) or immunogenic domain(s) orepitope(s) thereof.
 144. The isolated polynucleotide molecule of claim143, wherein one or more RSV glycoprotein gene(s) selected from F, G andSH is/are added, substituted or rearranged within said recombinant RSVgenome or antigenome to a position that is more promoter-proximalcompared to a wild type gene order position of said one or more RSVglycoprotein gene(s).
 145. The isolated polynucleotide molecule of claim144, wherein the RSV glycoprotein gene G is rearranged within saidrecombinant RSV genome or antigenome to a gene order position that ismore promoter-proximal compared to the wild type gene order position ofG.
 146. The isolated polynucleotide molecule of claim 145, wherein theRSV glycoprotein gene G is shifted to gene order position 1 within saidrecombinant RSV genome or antigenome.
 147. The isolated polynucleotidemolecule of claim 144, wherein the RSV glycoprotein gene F is rearrangedwithin said recombinant RSV genome or antigenome to a gene orderposition that is more promoter-proximal compared to the wild type geneorder position of F.
 148. The isolated polynucleotide molecule of claim147, wherein the RSV glycoprotein gene F is shifted to gene orderposition 1 within said recombinant RSV genome or antigenome.
 149. Theisolated polynucleotide molecule of claim 144, wherein both RSVglycoprotein genes G and F are rearranged within said recombinant RSVgenome or antigenome to gene order positions that are morepromoter-proximal compared to the wild type gene order positions of Gand F.
 150. The isolated polynucleotide molecule of claim 149, whereinthe RSV glycoprotein gene G is shifted to gene order position 1 and theRSV glycoprotein gene F is shifted to gene order position 2 within saidrecombinant RSV genome or antigenome.
 151. The isolated polynucleotidemolecule of claim 144, wherein one or more RSV NS1, NS2, SH, M2(ORF2),or G gene(s) or genome segment(s) thereof is/are deleted in therecombinant RSV genome or antigenome.
 152. The isolated polynucleotidemolecule of claim 144, wherein a displacement polynucleotide comprisinga RSV NS1 gene is deleted to form the recombinant RSV genome orantigenome.
 153. The isolated polynucleotide molecule of claim 144,wherein a displacement polynucleotide comprising a RSV NS2 gene isdeleted to form the recombinant RSV genome or antigenome.
 154. Theisolated polynucleotide molecule of claim 144 wherein a displacementpolynucleotide comprising a RSV SH gene is deleted to form therecombinant RSV genome or antigenome.
 155. The isolated polynucleotidemolecule of claim 154, wherein the RSV glycoprotein gene G is rearrangedwithin said recombinant RSV genome or antigenome to a gene orderposition that is more promoter-proximal compared to the wild type geneorder position of G.
 156. The isolated polynucleotide molecule of claim155, wherein the RSV glycoprotein gene G is shifted to gene orderposition 1 within said recombinant RSV genome or antigenome.
 157. Theisolated polynucleotide molecule of claim 154, wherein the RSVglycoprotein gene F is rearranged within said recombinant RSV genome orantigenome to a gene order position that is more promoter-proximalcompared to the wild type gene order position of F.
 158. The isolatedpolynucleotide molecule of claim 157, wherein the RSV glycoprotein geneF is shifted to gene order position 1 within said recombinant RSV genomeor antigenome.
 159. The isolated polynucleotide molecule of claim 158,which is F1/ΔSH.
 160. The isolated polynucleotide molecule of claim 154,wherein both RSV glycoprotein genes G and F are rearranged within saidrecombinant RSV genome or antigenome to gene order positions that aremore promoter-proximal compared to the wild type gene order positions ofG and F.
 161. The isolated polynucleotide molecule of claim 160, whereinthe RSV glycoprotein gene G is shifted to gene order position 1 and theRSV glycoprotein gene F is shifted to gene order position 2 within saidrecombinant RSV genome or antigenome.
 162. The isolated polynucleotidemolecule of claim 144, wherein the RSV SH and NS2 genes are both deletedto form the recombinant RSV genome or antigenome or antigenome.
 163. Theisolated polynucleotide molecule of claim 162, wherein both RSVglycoprotein genes G and F are rearranged within said recombinant RSVgenome or antigenome to gene order positions that are morepromoter-proximal compared to the wild type gene order positions of Gand F.
 164. The isolated polynucleotide molecule of claim 163, whereinthe RSV glycoprotein gene G is shifted to gene order position 1 and theRSV glycoprotein gene F is shifted to gene order position 2 within saidrecombinant RSV genome or antigenome.
 165. The isolated polynucleotidemolecule of claim 164, wherein the RSV SH, NS1 and NS2 genes are alldeleted to form the recombinant RSV genome or antigenome or antigenome.166. The isolated polynucleotide molecule of claim 165, wherein both RSVglycoprotein genes G and F are rearranged within said recombinant RSVgenome or antigenome to gene order positions that are morepromoter-proximal compared to the wild type gene order positions of Gand F.
 167. The isolated polynucleotide molecule of claim 166, whereinthe RSV glycoprotein gene G is shifted to gene order position 1 and theRSV glycoprotein gene F is shifted to gene order position 2 within saidrecombinant RSV genome or antigenome.
 168. The isolated polynucleotideof claim 124, wherein the recombinant genome or antigenome comprises apartial or complete human or bovine RSV background genome or antigenomecombined with one or more heterologous gene(s) and/or genome segment(s)from a different RSV to form a human-bovine chimeric genome orantigenome.
 169. The isolated polynucleotide of claim 168, wherein oneor both human RSV glycoprotein genes F and G is/are substituted toreplace one or both counterpart F and G glycoprotein genes in a partialbovine RSV background genome or antigenome.
 170. The isolatedpolynucleotide of claim 169, wherein both human RSV glycoprotein genes Fand G are substituted to replace counterpart F and G glycoprotein genesin the bovine RSV background genome or antigenome.
 171. The isolatedpolynucleotide of claim 168, wherein one or more human RSV glycoproteingenes selected from F, G and SH is/are added or substituted at aposition that is more promoter-proximal compared to a wild-type geneorder position of a counterpart gene or genome segment within a partialor complete bovine RSV background genome or antigenome.
 172. Theisolated polynucleotide of claim 171, wherein both human RSVglycoprotein genes G and F are substituted at gene order positions 1 and2, respectively, to replace counterpart G and F glycoprotein genesdeleted at wild type positions 7 and 8, respectively in a partial bovineRSV background genome or antigenome.
 173. The isolated polynucleotide ofclaim 124, wherein the recombinant genome or antigenome is furthermodified by addition or substitution of one or more additionalheterologous gene(s) or genome segment(s) from a human RSV within thepartial or complete bovine background genome or antigenome to increasegenetic stability or alter attenuation, reactogenicity or growth inculture of the recombinant virus.
 174. The isolated polynucleotidemolecule of claim 124, wherein the recombinant genome or antigenomeincorporates antigenic determinants from both subgroup A and subgroup Bhuman RSV.
 175. The isolated polynucleotide molecule of claim 124,wherein the recombinant genome or antigenome is further modified byincorporation of one or more attenuating mutations.
 176. The isolatedpolynucleotide molecule of claim 124, wherein the recombinant genome orantigenome is further modified by incorporation of a nucleotidemodification specifying a phenotypic change selected from a change ingrowth characteristics, attenuation, temperature-sensitivity,cold-adaptation, plaque size, host-range restriction, or a change inimmunogenicity.
 177. The isolated polynucleotide molecule of claim 176,wherein a SH, NS1, NS2, M2ORF2, or G gene is modified.
 178. The isolatedpolynucleotide molecule of claim 177, wherein the SH, NS 1, NS2, M2ORF2, or G gene is deleted in whole or in part or expression of the geneis ablated by introduction of one or more stop codons in an open readingframe of the gene.
 179. The isolated polynucleotide molecule of claim176, wherein the nucleotide modification comprises a nucleotidedeletion, insertion, addition or rearrangement of a cis-actingregulatory sequence of a selected RSV gene within the recombinant RSVgenome or antigenome.
 180. The isolated polynucleotide molecule of claim124, wherein said displacement polynucleotide(s) comprise(s) one or moredeletion(s) within a nontranslated sequence at the beginning or end ofan RSV open reading frame or in an intergenic region or 3′ leader or 5′trailer portion of the RSV genome.
 181. The isolated polynucleotidemolecule of claim 180, wherein said displacement polynucleotidescomprise or partial gene deletion.
 182. The isolated polynucleotidemolecule of claim 181, wherein said partial gene deletion is a partialdeletion of the SH gene.
 183. The isolated polynucleotide molecule ofclaim 182, wherein said partial deletion of the SH gene comprises adeletion within the SH downastream non-translated region.
 184. Theisolated polynucleotide molecule of claim 183, which is RSV 6120 havinga deletion of 112 nucleotides at positions 4499-4610 in the recombinantRSV antigenome.
 185. The isolated polynucleotide molecule of claim 124,wherein said displacement polynucleotide(s) is/are selected from one ormore region(s) of a downstream untranslated sequence of an RSV gene.186. The isolated polynucleotide molecule of claim 185, wherein saiddownstream untranslated sequence(s) is/are from NS1 (positions 519-563),NS2 (positions 1003-1086), P (positions 3073-3230), M (positions4033-4197), F(positions 7387-7539), and/or M2 (positions 8433-8490)genes.
 187. The isolated polynucleotide molecule of claim 124, whereinsaid displacement polynucleotide(s) is/are selected from one or moreregion(s) of a upstream untranslated sequence of an RSV gene.
 188. Theisolated polynucleotide molecule of claim 187, wherein said one or moreupstream untranslated sequences is/are from NS1 (positions 55-96), NS2(positions 606-624) and/or SH (positions 4231-4300).
 189. The isolatedpolynucleotide molecule of claim 124, wherein said displacementpolynucleotide comprises a deletion of nucleotides 4683 to 4685 of theRSV G gene.
 190. The isolated polynucleotide molecule of claim 124,wherein said displacement polynucleotide(s) is/are selected from one ormore RSV intergenic sequences.
 191. The isolated polynucleotide moleculeof claim 124, wherein said displacement polynucleotide(s) is/areselected from nucleotides within the RSV 5′ trailer region.
 192. Theisolated polynucleotide molecule of claim 191, wherein a portion of the5′ trailer region that immediately follows the L gene is reduced in sizeby 75 nucleotides, 100 nucleotides, 125 nucleotides, or more, leavingintact the 5′ genomic terminus.
 193. The isolated polynucleotidemolecule of claim 124, wherein said displacement polynucleotide(s)is/are selected from nucleotides within the RSV 3′ leader region. 194.The isolated polynucleotide molecule of claim 193, wherein a portion ofthe 3′ trailer region that excludes a core portion of the viral promoterlocated within the first 11 nucleotides of the 3′ leader is deleted.195. The isolated polynucleotide molecule of claim 124, wherein apartial or complete deletion from one or any combination of the RSV NS1,NS2, SH, F and/or M2 genes yields an adjustable reduction in genomelength of between 1-806 nucleotides.
 196. The isolated polynucleotidemolecule of claim 124, wherein a partial or complete deletion from oneor any combination of RSV intergenic regions yields an adjustablereduction in genome length of between 1-198 nucleotides.
 197. Theisolated polynucleotide molecule of claim 124, wherein a partial orcomplete deletion from one or any combination of RSV intergenic regionsyields an adjustable reduction in genome length of between 1-198nucleotides.
 198. A method for producing an infectious attenuatedrecombinant RSV particle from one or more isolated polynucleotidemolecules encoding said RSV, comprising: expressing in a cell orcell-free lysate an expression vector comprising an isolatedpolynucleotide comprising a recombinant RSV genome or antigenome havingone or more shifted RSV gene(s) or genome segment(s) within saidrecombinant genome or antigenome that is/are positionally shifted to amore promoter-proximal or promoter-distal position relative to aposition of said RSV gene(s) or genome segment(s) within a wild type RSVgenome or antigenome, and RSV N, P, L and RNA polymerase elongationfactor proteins.
 199. The method of claim 198, wherein the recombinantRSV genome or antigenome and the N, P, L and RNA polymerase elongationfactor proteins are expressed by two or more different expressionvectors.
 200. An isolated infectious chimeric respiratory syncytialvirus (RSV) comprising a major nucleocapsid (N) protein, a nucleocapsidphosphoprotein (P), a large polymerase protein (L), a RNA polymeraseelongation factor, and a partial or complete bovine RSV backgroundgenome or antigenome combined with a plurality of heterologous gene(s)and/or genome segment(s) of a human RSV selected from heterologousgene(s) and/or genome segment(s) of RSV NS1, NS2, M, SH, G, and/or F, toform a human-bovine chimeric RSV genome or antigenome.
 201. The isolatedinfectious RSV of claim 200, wherein both human NS1 and NS2 genes aresubstituted for their bovine counterpart NS1 and NS2 genes.
 202. Theisolated infectious RSV of claim 201, which is rBRSV/A2-NS1+2.
 203. Theisolated infectious RSV of claim 200, wherein human NS1, NS2, G, and Fare substituted for their bovine counterpart NS1, NS2, G and F genes.204. The isolated infectious RSV of claim 203, which isrBRSV/A2-NS1+2GF.
 205. The isolated infectious RSV of claim 200, whereinhuman M, SH, G, and F are substituted for their bovine counterpart M,SH, G and F genes.
 206. The isolated infectious RSV of claim 203, whichis rBRSV/A2-MSHGF.
 207. An isolated polynucleotide molecule comprising arecombinant RSV genome or antigenome comprising a partial or completebovine RSV background genome or antigenome combined with a plurality ofheterologous gene(s) and/or genome segment(s) of a human RSV selectedfrom heterologous gene(s) and/or genome segment(s) of RSV NS1, NS2, M,SH, G, and/or F genes, to form a human-bovine chimeric RSV genome orantigenome.